U.S. patent application number 12/739149 was filed with the patent office on 2010-11-25 for protein scaffolds.
This patent application is currently assigned to MEDIMMUNE, LLC. Invention is credited to Manuel Baca, Benoy Chacko, Jeffrey Swers, Herren Wu.
Application Number | 20100298541 12/739149 |
Document ID | / |
Family ID | 40591700 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100298541 |
Kind Code |
A1 |
Wu; Herren ; et al. |
November 25, 2010 |
Protein Scaffolds
Abstract
The invention provides protein scaffolds and methods of
preparing, screening, engineering and using such protein
scaffolds.
Inventors: |
Wu; Herren; (Boyds, MD)
; Baca; Manuel; (Gaithersburg, MD) ; Swers;
Jeffrey; (Rockville, MD) ; Chacko; Benoy;
(Gaithersburg, MD) |
Correspondence
Address: |
MEDIMMUNE, LLC;Patrick Scott Alban
ONE MEDIMMUNE WAY
GAITHERSBURG
MD
20878
US
|
Assignee: |
MEDIMMUNE, LLC
Gaithersburg
MD
|
Family ID: |
40591700 |
Appl. No.: |
12/739149 |
Filed: |
October 31, 2008 |
PCT Filed: |
October 31, 2008 |
PCT NO: |
PCT/US08/12398 |
371 Date: |
August 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60984209 |
Oct 31, 2007 |
|
|
|
Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 17/02 20180101; A61P 31/12 20180101; A61P 37/06 20180101; C12N
15/1044 20130101; A61P 19/08 20180101; C07K 2318/20 20130101; A61P
17/06 20180101; A61P 1/04 20180101; A61P 31/04 20180101; A61P 27/06
20180101; C07K 16/2878 20130101; A61P 7/06 20180101; A61P 37/08
20180101; C12N 15/1037 20130101; A61P 37/02 20180101; A61P 11/00
20180101; A61P 11/06 20180101; A61P 35/00 20180101; A61P 29/00
20180101; C07K 2319/00 20130101; A61P 3/04 20180101; A61P 17/14
20180101; A61P 3/10 20180101; A61P 9/10 20180101; A61P 27/02
20180101; A61P 35/02 20180101; C07K 14/78 20130101; A61P 31/10
20180101; G01N 33/566 20130101; A61P 1/00 20180101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/00 20060101
C07K014/00 |
Claims
1. A recombinant polypeptide scaffold comprising: I. seven beta
strand domains designated A, B, C, D, E, F, and G; II. linked to
six loop regions, wherein a loop region connects each beta strand
and is designated AB, BC, CD, DE, EF, and FG loops; III. wherein at
least one loop regions is a non-naturally occurring variant of the
cognate loop region in any of SEQ ID NOs: 1-32 or 68-88; and IV.
wherein at least one beta strand domain has at least 50%, at least
60%, at least 70%, at least 80%, or at least 90%, at least 95%, or
at least 99% homology to the cognate beta strand domain in any of
SEQ ID NOs: 1-32 or 68-88.
2. The scaffold of claim 1, wherein said beta strand domains
comprises the polypeptide sequence encoded by any of SEQ ID NOs:
1-32 or 68-88.
3. The scaffold of claim 1, wherein said beta strand domains
comprise: a. amino acid sequences for the A beta strand (SEQ ID
NO:228), B beta strand (SEQ ID NO:229), C beta strand (SEQ ID
NO:230), D beta strand (SEQ ID NO:231), E beta strand (SEQ ID
NO:232), F beta strand (SEQ ID NO:233) and G beta strand (SEQ ID
NO:234); or b. amino acid sequences for the A beta strand (SEQ ID
NO:235), B beta strand (SEQ ID NO:229), C beta strand (SEQ ID
NO:230), D beta strand (SEQ ID NO:236), E beta strand (SEQ ID
NO:232), F beta strand (SEQ ID NO:237) and G beta strand (SEQ ID
NO:234).
4. The scaffold of claim 3, wherein said BC loop comprises 9 amino
acids having a sequence of S-X-a-X-b-X-X-X-G, wherein X represents
any amino acid, wherein (a) represents proline or alanine and
wherein (b) represents alanine or glycine.
5. The scaffold of claim 3, wherein said BC loop comprises 11 amino
acids having a sequence of S-P-c-X-X-X-X-X-X T G, wherein X
represents any amino acid and wherein (c) represents proline,
serine or glycine.
6. The scaffold of claim 3, wherein said BC loop comprises 12 amino
acids having a sequence of A-d-P X-X-X-e-f-X-I-X-G, wherein X
represents any amino acid, wherein (d) represents proline,
glutamate or lysine, wherein (e) represents asparagine or glycine,
and wherein (f) represents serine or glycine.
7. The scaffold of claim 3, wherein said FG loop comprises 9 amino
acids having a sequence of X-a-X-X-G-X-X-X-S, wherein X represents
any amino acid and wherein (a) represents asparagine, threonine or
lysine.
8. The scaffold of claim 3, wherein said FG loop comprises 10 amino
acids having a sequence of X-a-X-X-X-X-b-N-P-A, wherein X
represents any amino acid, wherein (a) represents asparagine,
threonine or lysine and wherein (b) represents serine or
glycine.
9. The scaffold of claim 3, wherein said FG loop comprises 11 amino
acids having a sequence of X-a-X-X-G-X-X-S-N-P-A, wherein X
represents any amino acid, and wherein (a) represents asparagine,
threonine or lysine.
10. The scaffold of claim 3, wherein said DE loop comprises 6 amino
acids having a sequence of X-X-X-X-X-X, wherein X represents any
amino acid.
11. The scaffold of claim 3, wherein said AB loop comprises 7
residues having the sequence: K-X-X-X-X-X-a, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine, and
wherein (a) represents serine, threonine, alanine, or glycine.
12. The scaffold of claim 3, wherein said AB loop comprises 9
residues having the sequence: K-X-X-X-X-X-X-X-a, wherein X
represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, and wherein (a) represents serine, threonine,
alanine, or glycine.
13. The scaffold of claim 3, wherein said CD loop comprises 7, 8,
or 9 residues wherein each residue in the CD loop is randomized and
wherein each residue may be asparagine, aspartic acid, histidine,
tyrosine, isoleucine, valine, leucine, phenylalanine, threonine,
alanine, proline, or serine.
14. The scaffold of claim 3, wherein said EF loop comprises 8
residues having the sequence X-b-L-X-P-X-c-X, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine,
wherein (b) represents asparagine, lysine, arginine, aspartic acid,
glutamic acid, or glycine, and wherein (c) represents isoleucine,
threonine, serine, valine, alanine, or glycine.
15. The scaffold of claim 1, wherein said scaffold further
comprises at least one disulfide bond to link any two of said seven
beta strand domains.
16. The scaffold of claim 15, wherein said disulfide bond forms a
link between: a. the A beta strand and B beta strand; b. the D beta
strand and E beta strand; c. the F beta strand and G beta strand;
or d. the C beta strand and F beta strand.
17. The scaffold of claim 15, wherein said scaffold further
comprises at least two disulfide bonds to link any of said seven
beta strand domains.
18. The scaffold of claim 17, wherein said disulfide bonds form a
first link between the F beta strand and the G beta strand, and a
second link between the C beta strand and F beta strand.
19. The scaffold of claim 1, wherein said scaffold further
comprises at least one disulfide bond in a loop region.
20. The scaffold of claim 15, wherein said scaffold comprises the
sequence corresponding to Tn3.sup.SS1(SEQ ID NO:64),
Tn3.sup.SS2(SEQ ID NO:210), Tn3.sup.SS3(SEQ ID NO:65), or
Tn3.sup.SS4(SEQ ID NO:66).
21-96. (canceled)
Description
1. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. provisional
application No. 60/984,209, filed Oct. 31, 2007, which is
incorporated by reference in its entirety.
2. FIELD OF THE INVENTION
[0002] This invention relates to protein scaffolds that
specifically bind a target and methods of making, screening and
using such scaffolds.
3. BACKGROUND OF THE INVENTION
[0003] This invention relates to protein scaffolds useful, for
example, for the generation of products having novel binding
characteristics.
[0004] Proteins having relatively defined three-dimensional
structures, commonly referred to as protein scaffolds, may be used
as reagents for the design of engineered products. These scaffolds
typically contain one or more regions which are amenable to
specific or random sequence variation, and such sequence
randomization is often carried out to produce libraries of proteins
from which desired products may be selected. One particular area in
which such scaffolds are useful is the field of antibody mimetic
design.
[0005] While therapeutic antibodies are known with some successful
examples on the market (HERCEPTIN.RTM., AVASTIN.RTM.,
SYNAGIS.RTM.), there is a growing interest in generating antibody
fragments as therapeutic proteins. The advantages are the ease of
manipulation by molecular biology techniques in order to obtain
desired binding characteristics, the ability to express such
fragments in microbial systems, and the expectation that antibody
fragments will have better tissue penetration than full-length
antibodies. One example is REOPRO.RTM..
[0006] In addition, there have been efforts to develop small,
non-antibody therapeutics, i.e., antibody mimetics, in order to
capitalize on the advantages of antibodies and antibody fragments,
such as high affinity binding of targets and low immunogenicity and
toxicity, while avoiding some of the shortfalls, such as the
requirement for intradomain disulfide bonds which require proper
refolding, and the tendency for antibody fragments to aggregate and
be less stable than full-length IgGs. One example is a "minibody"
scaffold, which is related to the immunoglobulin fold, which is
designed by deleting three beta strands from a heavy chain variable
domain of a monoclonal antibody (Tramontano et al., J. Mol.
Recognit. 7:9, 1994). This protein includes 61 residues and can be
used to present two hypervariable loops, much like complementarity
determining regions (CDRs) in antibodies. These two loops have been
randomized and products selected for antigen binding, but thus far
the framework appears to have somewhat limited utility due to
solubility problems. Another framework used to display loops has
been tendamistat, a small protein inhibitor of .alpha.-amylase,
which contains a 74 residue, six-strand beta sheet sandwich held
together by two disulfide bonds and forms 3 CDR-like loops
(McConnell and Hoess, J. Mol. Biol. 250:460, 1995).
[0007] Other proteins have been tested as frameworks and have been
used to display randomized residues on alpha helical surfaces (Nord
et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Protein Eng.
8:601, 1995), loops between alpha helices in alpha helix bundles
(Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and
loops constrained by disulfide bridges, such as those of the small
protease inhibitors (Markland et al., Biochemistry 35:8045, 1996;
Markland et al., Biochemistry 35:8058, 1996; Rottgen and Collins,
Gene 164:243, 1995; Wang et al., J. Biol. Chem. 270:12250,
1995).
[0008] Thus, there is a need to develop small, stable, artificial
antibody-like molecules for a variety of therapeutic and diagnostic
applications.
[0009] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
4. SUMMARY OF THE INVENTION
[0010] The present invention provides a family of recombinant,
non-naturally occurring protein scaffolds capable of binding any
compound of interest. In particular, the proteins described herein
may be used to display defined loops which are analogous to the
complementarity-determining regions ("CDRs") of an antibody
variable region. These loops maybe subjected to randomization or
restricted evolution to generate diversity required to bind a
multitude of target compounds. The proteins may be assembled into
multispecific scaffolds capable of binding different targets.
[0011] The invention provides recombinant, non-naturally occurring
polypeptide scaffolds (herein after known as "scaffolds of the
invention") comprising, a plurality of beta strand domains linked
to a plurality of loop region sequences derived from a naturally
occurring protein sequence, wherein one or more of said loop region
sequences vary by deletion, substitution or addition by at least
one amino acid from the corresponding loop sequences in the
naturally occurring protein sequence, and wherein the beta strand
domains of the polypeptide scaffold have at least 50% homology to
the corresponding domain sequences of a naturally occurring protein
sequence. In some embodiments, the naturally occurring sequence is
the protein sequence corresponding to human tenascin C. In
particular, these scaffolds include, the third FnIII domain of
tenascin C (also known as the "Tn3" domain). In specific
embodiments, scaffolds of the invention comprise an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1, 5-32,
64-67, and 210. In other specific embodiments, scaffolds of the
invention may be encoded by a nucleic acid comprising a sequence
selected from the group consisting of SEQ ID NOs:33-59.
[0012] In other embodiments, the naturally occurring sequence
corresponds to a predicted Tn3 structural motif, such as those
derived from a thermophilic organism, for example but not limited
to, Archaeoglobus fulgidus, Staphylothermus marinus, Sulfolobus
acidocaldarius, Sulfolobus solfataricus, and Sulfolobus
tokodaii.
[0013] In specific embodiments, scaffolds of the invention comprise
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2-4, 68-88, and 210. In other specific embodiments, scaffolds
of the invention may be encoded by a nucleic acid comprising a
sequence selected from the group consisting of SEQ ID
NOs:89-98.
[0014] In another aspect of the invention, the scaffolds of the
invention also include disulfide-stabilized scaffolds. The
disulfide-stabilized scaffolds exhibit enhanced stability as
measured by thermal tolerance, resistance to chaotropic
denaturation and protease treatment.
[0015] The scaffolds of the invention are engineered to bind
targets of interest, as described herein. Such binding may, for
example, exhibit an affinity of at least 100 .mu.M.
[0016] The invention also provides multimeric scaffolds comprising
at least two scaffolds of the invention (hereinafter known as
"multimeric scaffolds of the invention"). In some embodiments, the
multimeric scaffolds of the invention comprise at least two
scaffolds linked, for example, but not limited to, a dimerization
domain, an amino acid linker, a disulfide bond, a chemical
crosslink, and IgG molecule or fragment thereof, or an Fc
region.
[0017] The invention also provides polypeptide display libraries
(hereinafter referred to as "libraries of the invention")
comprising a plurality of scaffolds of the invention. The libraries
of the invention are useful for capturing and identifying target
binding scaffolds to build multimeric scaffolds.
[0018] In another aspect the invention also provides isolated
nucleic acid molecules encoding the scaffolds and libraries of the
invention.
[0019] The invention also provides methods of making, using,
screening, optimizing, and engineering the scaffolds and libraries
of the invention.
[0020] In yet another aspect, the invention also provides
pharmaceutical compositions comprising the scaffolds of the
invention.
[0021] The invention also provides methods of treating, preventing,
ameliorating, detecting, diagnosing, or monitoring a disease or
symptoms thereof, as described herein, in a patient by
administering therapeutically effective amounts of the scaffolds of
the invention and/or pharmaceutical compositions comprising the
scaffolds of the invention.
[0022] In specific embodiments, the invention provides TRAIL-R2
specific binders which are useful for preventing ameliorating,
detecting, diagnosing, or monitoring diseases, such as but not
limited to cancer. In other specific embodiments, TRAIL-R2 specific
binding scaffolds of the invention are useful for the treatment of
cancers in which cancer cells express TRAIL-R2. In some
embodiments, cancers may include, but are not limited to, lung
cancer, non-Hodgkin's lymphoma, gastrointestinal cancer, renal
cancer, ovarian cancer, liver cancer, stomach cancer, bladder
cancer, breast cancer, colon cancer, colorectal cancer, pancreatic
cancer, liver cancer, prostate cancer, and melanoma.
5. BRIEF DESCRIPTION OF THE FIGURES
[0023] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments on the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0024] FIG. 1. Recombinant total cell expression of scaffolds.
Presented here is a Coomassie stained PAGE gel of various
recombinant lysates from E. coli cultures expressing protein
scaffolds. Highly expressed scaffolds are indicated with an
asterisk (*).
[0025] FIG. 2. Expression and purification of a Tn3 based scaffold.
Presented here is a Coomassie stained PAGE gel documenting the Tn3
based scaffold after recombinant expression and purification. The
Tn3 scaffold was readily purified to homogeneity.
[0026] FIG. 3A. Melting temperature determination for a Tn3 based
scaffold. The graph illustrates the thermal melting curve
determination of the Tn3 based scaffold as measured by differential
scanning calorimetry. The T.sub.m was determined to be about
45.degree. C. at pH 7.0.
[0027] FIG. 3B. Urea denaturation profile of a Tn3 based scaffold.
The graph represents the denaturation profile of a Tn3 based
scaffold in various concentrations of urea at pH 7.5. The change in
intrinsic fluorescence of the molecules was used as a measure of
unfolding of the molecule. For the Tn3 scaffold, unfolding results
in an increase in fluorescence intensity and shift to a higher
wavelength. As demonstrated in the figure, the molecule is folded
at low concentrations of urea (less than or equal to 1M) and became
denatured at higher concentrations of urea. The molarity at which
50% of the molecules are unfolded was calculated to be about
2M.
[0028] FIG. 3C. A Tn3 scaffold exists in a monomeric state. The
graph represents the results of a size exclusion chromatography
analysis of a purified Tn3 scaffold to determine the relative
proportions of contaminating fragments and/or higher order
structures. As represented by the percentages on the graph, over
97% of the scaffold eluted in monomeric form (determined to be
around 11 kDa by an online light scattering detector) while only a
small fraction (around 3%) eluted at a peak of about 21 kDa,
possibly as a dimer.
[0029] FIG. 4. The BC loops of scaffolds demonstrate loop length
diversity. The graph represents the length diversity of the BC loop
of various Tn3 related protein scaffolds. (A) represents the loop
length diversity exhibited by sequences derived from a subset of
Tn3 related scaffolds from the PDB database (51 sequences). (B)
represents the loop length diversity exhibited by sequences derived
from the Swiss-prot database (397 sequences). As presented, the BC
loop of various Tn3 related scaffolds exhibit lengths from 7-26
amino acid residues. According to the scheme used herein, the
length of the BC loops in Tn3 is 9 residues.
[0030] FIG. 5. The FG loops of scaffolds demonstrate loop length
diversity. The graph represents the length diversity of the FG
loops of various Tn3 related protein scaffolds. The figure depicts
the FG loop length diversity exhibited by sequences derived from a
subset of Tn3 related scaffolds from the PDB database. As
presented, the FG loop of Tn3 scaffolds exhibit lengths of 6-18
amino acid residues. According to the scheme used herein, the
length of the FG loop in Tn3 is 10 residues.
[0031] FIG. 6. The DE loops of scaffolds demonstrate loop length
diversity. The graph represents the length diversity of the DE
loops of various Tn3 protein scaffolds. The figure depicts the DE
loop length diversity exhibited by sequences derived from a subset
of Tn3 related scaffolds from the PDB database. As presented, the
DE loop of Tn3 related scaffolds exhibit lengths from about 4 to
about 17 amino acid residues. According to the scheme used herein,
the length of the DE loop in Tn3 is 6 residues.
[0032] FIG. 7A. The 9 amino acid residue BC loops of scaffolds
demonstrate sequence diversity. The graph represents the sequence
diversity exhibited by 9 amino acid residue BC loops from the
Swiss-Prot database (73 sequences). Using the alignment tool,
Weblogo, the relative incidence of an amino acid occurring at a
particular position in a 9 amino acid long BC loop is represented
by the size of the single letter code above the particular
position. For example, at position 3 of the analyzed BC loops of 9
amino acid residues, the most prevalent amino acid is Proline (P),
followed by Alanine (A), followed by Valine (V).
[0033] FIG. 7B. The 12 amino acid residue BC loops of scaffolds
demonstrate sequence diversity. The graph represents the sequence
diversity exhibited by 12 amino acid residue BC loops from the
Swiss-Prot data set (99 sequences). Using the alignment tool,
Weblogo, the relative incidence of an amino acid occurring at a
particular position in a 12 amino acid long BC loop is represented
by the size of the single letter code above the particular
position. For example, at position 3 of the analyzed BC loops of 12
amino acid residues, the most prevalent amino acid is Proline (P),
followed by Glycine (G), followed by Glutamine (Q).
[0034] FIG. 8. The FG loops of scaffolds demonstrate sequence
diversity. The graph represents the sequence diversity exhibited by
FG loops from all of the lengths from the Swiss-Prot data set (393
sequences). Using the alignment tool, Weblogo, the relative
incidence of an amino acid occurring at a particular position in
the FG loop is represented by the size of the single letter code
above the particular position. For example, at position 2 of the
analyzed FG loops, the most prevalent amino acid is Asparagine (N),
followed by Threonine (T), followed by Lysine (K).
[0035] FIG. 9A. Expression and purification of a SYNAGIS.RTM.
specific Tn3 scaffold. Presented here is a Coomassie stained PAGE
gel documenting the SYNAGIS.RTM. specific Tn3 scaffold (SynBP01)
after recombinant expression and purification. The SynBP01 scaffold
was readily purified to homogeneity.
[0036] FIG. 9B. The K.sub.D, determination of SYNAGIS.RTM. specific
binding scaffold. The tracing represents an experiment
characterizing the specific binding affinity for the SynBP01
scaffold for SYNAGIS.RTM. by a BIACORE.RTM. assay. Using
immobilized SYNAGIS.RTM.and mobile phase SynBP01 at various
concentrations it was determined that the binding affinity
(K.sub.D) was about 16 .mu.M.
[0037] FIG. 9C. The SYNAGIS.RTM. specific binding scaffold exhibits
a similar stability to the base scaffold. A SYNAGIS.RTM. specific
binding scaffold was identified from a library of scaffolds as
prepared in Example 3. Presented here are the results from a Urea
denaturation experiment in which the intrinsic fluorescence
measures the unfolding of the proteins. In this experiment, the Tn3
scaffold (WT) and SYNAGIS.RTM. specific binding scaffold SynBP01
(Al) exhibited very similar denaturation profiles with increasing
Urea concentrations.
[0038] FIG. 9D. Binding scaffolds engage the target antibody
SYNAGIS.RTM., bivalently. The tracing represents an experiment
characterizing the engagement of two scaffolds on an immobilized
molecule in a BIAcore assay format. In this experiment, immobilized
SYNAGIS.RTM. is overlaid with a either a solution of 1 .mu.M
SYNAGIS.RTM. specific protein scaffold (blue tracing) or a solution
of 1 .mu.M SYNAGIS.RTM. specific protein scaffold+0.19 .mu.M
crosslinking mAb (red tracing). The graph depicts the differential
binding characteristics exhibited by monomer scaffolds or
crosslinked scaffolds.
[0039] FIG. 10A. Sequence alignment of naturally occurring
disulfide containing Tn3 structural motifs. The sequence alignment
presented outlines the position of cysteine residues in 21
naturally occurring disulfide containing Tn3 related structural
motifs in an effort to determine candidate positions for stability
engineering. Like-colored Cys residues within individual Tn3
sequences are linked by a disulfide bond. The sequence of Tn3
(Iten.pro) is included in the alignment to facilitate the
identification of residues and positions in Tn3 corresponding to
cysteine residues in disulfide-containing scaffolds.
[0040] FIG. 10B. Targeted disulfide engineering to increase
scaffold stability. The graphic depicts the potential disulfide
locations to be engineered into the scaffold sequence. In an effort
to increase stability, the four depicted disulfide locations were
individually engineered into the scaffold. The resultant
recombinant scaffolds were termed Tn3.sup.SS1, Tn3.sup.SS2,
Tn3.sup.SS3, and Tn3.sup.SS4 respectfully.
[0041] FIG. 10C. RP-HPLC tracing of selected purified disulfide
engineered scaffolds. Depicted in the graphic are the reverse-phase
HPLC chromatograms of (i) Tn3.sup.SS3 and (ii) Tn3.sup.SS4 after
purification, following reduction, and after refolding to form
disulfide bonds. The chromatograms demonstrated that the cysteines
contained within the scaffolds are only partially oxidized
following protein purification (upper tracing) are completely
reduced by treatment with DTT (middle tracing) and completely
oxidized to disulfides after refolding (lower tracing).
[0042] FIG. 10D. Expression and characterization of disulfide
engineered scaffolds. The figure depicts the results of a
polyacrylamide gel electrophoresis analysis of 4 disulfide
engineered scaffolds presented in FIG. 10B. Samples of purified and
refolded constructs Tn3 (WT), Tn3.sup.SS1 (1), Tn3.sup.SS2 (2),
Tn3.sup.SS3 (3), Tn3.sup.SS4 (4) were analyzed under reducing and
non-reducing conditions to evaluate the predicted formation of
disulfide bonds. As depicted, the Tn3.sup.SS2 forms a disulfide
linked dimer. The other constructs, (Tn3, Tn3.sup.SS1, Tn3.sup.SS3
and Tn3.sup.SS4) do not form dimers in response to the incorporated
cysteine residues as the purified and refolded disulfide containing
scaffolds migrate similarly under reducing and non-reducing
conditions.
[0043] FIG. 10E. Urea denaturation profile of the single disulfide
engineered scaffolds. The graph depicts the results of a urea
denaturation study of selected disulfide containing scaffolds
outlined in panel A Tn3.sup.SS1(SS1), Tn3.sup.SS3(SS3), and
Tn3.sup.SS4(SS4) as well as Tn3 (WT) scaffold. In this experiment
the various purified and refolded scaffolds are exposed to
increasing levels of urea. As a measure of unfolding, the relative
fluorescence is monitored. As a protein unfolds or becomes less
stable, the relative fluorescence increases. As presented in the
panel, the Tn3.sup.SS1 containing scaffold exhibits a higher
sensitivity to urea compared to the Tn3 scaffold and is therefore
less stable. Also, scaffolds Tn3.sup.SS3 and Tn3.sup.SS4 exhibit
greater resistance to urea mediated denaturation as compared to Tn3
and are therefore more stable. The scaffold Tn3.sup.SS4 exhibited
the greatest resistance to urea mediated denaturation of the
scaffolds tested in this experiment.
[0044] FIG. 10F. The Tn3.sup.SS4 disulfide engineered scaffold
exhibits increased stability as compared to the Tn3 scaffold. The
graph depicts the results from a guanidine hydrochloride
denaturation assay comparing the stability of the Tn3 scaffold
(circles) to the disulfide containing scaffold, Tn3.sup.SS4
(squares). As presented in the graph, the Tn3.sup.SS4 scaffold
exhibited a greater resistance to guanidine mediated denaturation,
and therefore, was more stable, as compared to the Tn3
scaffold.
[0045] FIG. 10G. The Tn3.sup.SS4 scaffold exhibits a high level of
protease resistance compared to the Tn3 scaffold. The panel depicts
the results from a protease sensitivity assay comparing the
stability of the Tn3 scaffold to the Tn3.sup.SS4 containing
scaffold. In this experiment, the relative protease resistance
correlates with the protein stability. For the Tn3 scaffold,
incubation for as little as ten minutes with thermolysin results in
degradation. After one hour of incubation in thermolysin, the Tn3
scaffold is completely degraded. The Tn3.sup.SS4 containing
scaffold exhibited thermolysin resistance over the entire 16 hour
time course suggesting a greater stability than the Tn3
scaffold.
[0046] FIG. 10H. The Tn3.sup.SS4 disulfide engineered scaffold
exhibits a high melting temperature (Tm). The graph depicts the
results from a thermal stability study comparing the Tn3 scaffold
(dotted line) to the disulfide containing Tn3.sup.SS4 scaffold
(solid line). As presented in the melt curve, the Tn3.sup.SS4
scaffold exhibited a higher melting temperature (about 71.degree.
C.) than the Tn3 scaffold (about 45.degree. C., See also FIG.
3A).
[0047] FIG. 10I. The Tn3.sup.SS4 disulfide engineered scaffold
exists in a monomeric state. The figure depicts a tracing from a
Size exclusion chromatography/multiple-angle light scattering
(SEC-MALS) analysis of the Tn3.sup.SS4 scaffold. The data
demonstrates that the purified Tn3.sup.SS4 scaffold existed in a
monomeric state, determined to be about 11 kDa by an online light
scattering detector.
[0048] FIG. 10J. RP-HPLC chromatograms of the purified
Tn3.sup.SS3+4 disulfide engineered scaffolds. Depicted in the
graphic are the reverse-phase HPLC chromatograms of Tn3.sup.SS3+4
after purification, following reduction, and after refolding to
form disulfide bonds. The tracings demonstrated that the cysteines
contained within the scaffolds are only partially oxidized
following protein purification (upper tracing) are completely
reduced by treatment with DTT (middle tracing) and are completely
oxidized to two disulfide bonds after refolding (lower
tracing).
[0049] FIG. 10K. The dual disulfide containing scaffold
Tn3.sup.SS3+4 exhibits an elevated level of stability. The graph
depicts the results from a guanidine hydrochloride denaturation
assay comparing the stability of the Tn3 (WT) scaffold (circles) to
the single disulfide containing scaffold, Tn3.sup.SS4 (squares) and
to the double disulfide containing scaffold, Tn3.sup.SS3+4
(triangles). As presented in the graph, the refolded double
disulfide scaffold, Tn3.sup.SS3+4 exhibited a greater resistance to
guanidine mediated denaturation, and therefore more stable, as
compared to the single refolded disulfide containing scaffold,
Tn3.sup.SS4 as well as the Tn3 scaffold.
[0050] FIG. 11A. Expression and purification of scaffolds from
hyperthermophilic organisms. Presented here are Coomassie-stained
PAGE gels documenting the expression and purification of predicted
scaffolds (Tn3 structural motifs) from various hyperthermophilic
bacteria. All of the predicted scaffolds were expressed and
purified to homogeneity. In the figure, T represents total cell
lysate, S represents soluble lysate fraction and P represents
purified protein.
[0051] FIG. 11B. A scaffold from Staphylothermus marinus exhibits a
high level of thermal stability. The graph depicts the thermal
stability of a scaffold from Staphylothermus marinus. The putative
scaffold was recombinantly expressed, purified, and subjected to
the thermal stability test. As presented in the graph, the scaffold
from Staphylothermus marinus exhibits a high melting temperature
(around 83.degree. C. at pH 7.0).
[0052] FIG. 11C. A scaffold from Sulfolobus tokodaii exhibits a
high level of stability. The panel depicts the thermal stability of
a scaffold from Sulfolobus tokodaii. The putative scaffold was
recombinantly expressed, purified, and subjected to the thermal
stability test in a differential scanning calorimeter. As presented
in the graph, the scaffold from Sulfolobus tokodaii exhibits a high
melting temperature (around 98.degree. C.) at pH 3.0. The scaffold
also exhibits a high level of stability at pH 7.0, however, it
aggregates and falls out of solution at temperatures greater than
75.degree. C.
[0053] FIG. 11D. A scaffold from Staphylothermus marinus exhibits a
high level of stability. The graph depicts the results from a
guanidine hydrochloride denaturation assay demonstrating the high
protein stability of a scaffold from Staphylothermus marinus. As
presented in the graph, a high concentration of guanidine
hydrochloride are required to unfold the scaffold (50% of the
molecules are unfolded at a guanidine concentration of 5.0 M),
which exemplified high stability.
[0054] FIG. 11E. A scaffold from Sulfolobus tokodaii exhibits a
high level of stability. The panel depicts the results from a
guanidine hydrochloride denaturation assay demonstrating the high
protein stability of a scaffold from Sulfolobus tokodaii. Protein
unfolding is correlated with an increase in relative fluorescence
of the molecule. As presented in the graph, a high concentration of
guanidine-HCl is required to unfold the scaffold at either pH 7.0
(circles) or at pH 3.0 (squares), which exemplified high
stability.
[0055] FIG. 11F. A scaffold from Staphylothermus marinus exhibits a
high level of protease resistance. The graph depicts the results
from a protease sensitivity assay measuring the stability of a
scaffold from Staphylothermus marinus. In this experiment, the
relative protease resistance correlates with the protein stability.
For the scaffold, incubation for ten minutes with thermolysin
results in a small level of degradation which remains stable over
time. The Staphylothermus marinus scaffold exhibited thermolysin
resistance over the entire 16 hour time course.
[0056] FIG. 11G. A scaffold from Sulfolobus tokodaii exhibits a
high level of protease resistance. The panel depicts the results
from a protease sensitivity assay measuring the stability of a
scaffold from Sulfolobus tokodaii. In this experiment, the relative
protease resistance correlates with the protein stability. The
Sulfolobus tokodaii scaffold exhibited thermolysin resistance over
the entire 16 hour time course.
[0057] FIG. 11H. A scaffold from Staphylothermus marinus is
purified from a host cell. The panel depicts one step in the
purification of the Staphylothermus marinus scaffold. Due to the
high level of stability exhibited by this scaffold, it is possible
to heat the crude E. coli lysate containing the recombinant
scaffold to remove the bulk of the host cell proteins while
retaining the scaffold. Lane 1 represents the crude lysate prior to
heat treatment. Coomassie staining of a PAGE gel containing the
crude lysate demonstrates that the candidate scaffold was present.
Heat treatment of the crude lysate at 70.degree. C. for 15 minutes
resulted in a loss of much of the host cell proteins while the
scaffold remains intact (Lane 2).
[0058] FIG. 11I. A scaffold from Staphylothermus marinus is
purified from a host cell. The panel depicts one step in the
purification of the Staphylothermus marinus scaffold. Due to the
high level of stability exhibited by this scaffold, it is possible
to treat the crude E. coli lysate containing the recombinant
scaffold with thermolysin to degrade the bulk of the host cell
proteins while retaining the scaffold. Lane 1 represents the crude
lysate prior to protease treatment. Coomassie staining of a PAGE
gel containing the crude lysate demonstrates that the scaffold is
present. Protease treatment of the crude lysate at 55.degree. C.
for 45 minutes resulted in a loss of much of the host cell proteins
while the scaffold remains intact (Lane 2).
[0059] FIG. 11J. A scaffold from Sulfolobus tokodaii is purified
from a host cell. The panel depicts the purification of the
Sulfolobus tokodaii scaffold. Due to the high level of stability
exhibited by this scaffold, it is possible to incubate the crude
lysate containing the recombinant scaffold at pH 3.0 and raise the
temperature to 70.degree. C. for 15 min to remove the majority of
the host cell proteins while retaining the soluble scaffold. Lane 1
represents the crude E. coli lysate prior to acidification or heat
treatment. Lane 2 represents the soluble protein remaining after
lowering the pH of the crude lysate to 3.0. Lane 3 represents the
soluble protein after lowering the pH to 3.0 and incubation at
70.degree. C. for 15 min. The scaffold was resistant and remained
in solution through these treatments.
[0060] FIG. 12. Two loops are required for binding of SynBP01 to
SYNAGIS.RTM.. This panel depicts the binding of SynBP01 and
variants thereof to plate bound SYNAGIS.RTM.. In this experiment,
the BC and FG loops of SynBP01 were transferred to a wild-type
scaffold to create single loop variants of SynBP01. These variants
(designated BC only and FG only) were subjected to an ELISA based
binding assay in which they did not exhibit binding to the plate
bound SYNAGIS.RTM.. As a control, SynBP01 binding in the ELISA
assay format is presented. In addition, the SS4 disulfide mutation
was overlaid on SynBP01. This variant, designated SynBP01 SS4 did
not exhibit any binding to plate bound SYNAGIS.RTM. in the ELISA
format.
[0061] FIG. 13. Binding affinity determination of the TRAIL-R2
specific clone 5E5 as measured by BIAcore. This panel depicts an
affinity determination of clone 5E5 for chip bound TRAIL-R2. The
K.sub.d was estimated to be approximately 700 nM.
[0062] FIG. 14. Competitive binding exhibited by multiple TRAIL-R2
specific clones. Depicted in this panel are the results from a
competitive binding experiment of various TRAIL-R2 specific
scaffolds. Two TRAIL-R2 specific clones, 5E5 and 7G11 were tested
for competitive binding against other TRAIL-R2 specific clones
displayed on phage. 5E5 competes with itself and all other clones
except 2H6 and 7G11. 7G11 only competes with itself and 2H6. This
data indicates that there are two groups of clones recognizing two
different epitopes on TRAIL-R2.
[0063] FIG. 15. Competition of various TRAIL-R2 specific clones.
Depicted in this panel are the results from a competitive binding
experiment of various TRAIL-R2 specific scaffolds. Five TRAIL-R2
specific clones, 1E3, 1G11, 2B4, 1C12 and 2D3 were tested for
competitive binding against other TRAIL-R2 specific clones
displayed on phage. 1E3, 1C12 and 2D3 competed with most of the
phage displayed clones except for 8B3 and 7G11. Soluble 1G11 did
not compete with any of the phage displayed clones, and 2B4 showed
little to moderate inhibition in most cases. This data suggests
that 1E3, 1C12 and 2D3 recognize the same epitope on TRAIL-R2.
[0064] FIG. 16. TRAIL-R2 binders inhibits cell viability. Depicted
in panel is the sensitivity of the Colo-205 cell line to the
treatment of 5E5, 7G11, 1C12, 2D3 and 1E3 when complexed with
anti-HIS tag antibody and anti-mouse IgG in the molar ratio of
2:1:0.5. The percent viable cells was obtained by expressing the
assay signal for treatment with TRAIL-R2 binding clones as a
percentage of the signal obtained for cells treated with a control
Tn3 which does not bind TRAIL-R2.
[0065] FIG. 17A. Diagram representing examples of Multivalent
Tn3-fusion proteins. The bivalent construct represents a Tn3 fused
to an antibody Fc region whereas the tetravalent construct fuses
the Tn3 to heavy chain constant regions and/or the light chain
Ckappa region.
[0066] FIG. 17B. Diagram showing linkage of protein modules in
Multivalent Tn3-fusion proteins. i) In the Fc fusion construct, the
N-terminal Tn3 module is linked to the Fc region of IgG1 via a
peptide linker and hinge regions of IgG1. The linker sequence can
vary in length and composition, but was a -GA- motif in Example 16.
ii) In the Ckappa fusion construct, the N-terminal Tn3 module is
linked to the constant region of an antibody kappa light chain via
a peptide linker. The linker sequence can vary in length and
composition, but was a -GGGTPT- motif in Example 16. iii) In the
IGHG1 fusion construct, the N-terminal Tn3 module is linked to the
constant region of an IgG1 heavy chain via a peptide linker. The
linker sequence can vary in length and composition, but was a
-GGGTPT- motif in Example 16.
[0067] FIG. 18. Expression of multivalent Tn3 constructs.
Represented in the figure is a reducing SDS-PAGE showing the
relative sizes of bivalent Tn3 constructs (samples 1-3), and
tetravalent Tn3 constructs (samples 4-7) after protein A
purification. Sample numbers correspond to those shown in Table 10.
All constructs expressed exhibit the predicted sizes by this
analysis.
[0068] FIG. 19. Binding affinity of Tn3 monomers and multimers.
Depicted in this panel are sensorgrams from a Biosensor binding
experiment using mono-, bi- and tetravalent 1C12 and D1 Tn3
constructs. TRAIL-R2-Fc fusion protein was immobilized on a sensor
chip while sample proteins were injected over the chip. "tetra"
refers to the tetravalent C.kappa./IGHG1 fusion constructs. The
1C12 constructs show improved binding to TRAIL-R2 as a function of
valency, while the control D1 contstructs do no exhibit
binding.
[0069] FIG. 20. Effects of Bivalent Tn3 constructs targeting
TRAIL-R2. Depicted in this panel are the growth inhibition curves
of H2122 cells that were treated with (A) TRAIL-R2 specific Tn3
binder 1C12 in bivalent form or a (B) non-specific Tn3 binder in
bivalent form with or without the addition of a crosslinking
antibody (anti-human Fc). Relative to the control non-binder, the
TRAIL-R2 specific binder exhibited mild activity in the assay,
which decreased in the presence of the secondary antibody.
[0070] FIG. 21. Effects of Tetravalent Tn3 constructs targeting
TRAIL-R2. Depicted in this panel are the growth inhibition curves
of H2122 cells that were treated with (A) TRAIL-R2 specific Tn3
binder 1C12 in monospecific tetravalent form or a (B) non-specific
Tn3 binder in monospecific tetravalent form with or without the
addition of a crosslinking antibody (anti-human Fc). Relative to
the control non-binder, the TRAIL-R2 specific binder exhibited
activity in the assay, which decreased slightly in the presence of
the secondary antibody.
[0071] FIG. 22. Effects of Multispecific Tetravalent Tn3 constructs
targeting TRAIL-R2. Depicted in this panel are the growth
inhibition curves of H2122 cells that were treated with (A)
TRAIL-R2 specific Tn3 binder 1C12 and 2D3 in a bispecific
tetravalent form (1C12 fused to the constant region of IgG1 heavy
chain, 2D3 fused to the Ckappa domain) or a (B) TRAIL-R2 specific
binder 2D3 and 1C12 in a bispecific tetravalent form (2D3 fused to
the constant region of IgG1 heavy chain, 1C12 fused to the Ckappa
domain) with or without the addition of a crosslinking antibody
(anti-human Fc). Relative to the control non-binder (shown in FIG.
21), the TRAIL-R2 specific binders exhibited activity in the assay,
which increased in the presence of the secondary antibody.
[0072] FIG. 23: Design of pSec constructs. (A) represents the amino
acid sequence for the open reading frame in pSec-oppA-Tn3. The oppA
signal peptide (underlined) SEQ ID NO:227 is cleaved after
secretion into the periplasmic space. The penultimate residue
within the oppA signal (italicized) was mutated to methionine in
the pSec-oppA(L25M)-Tn3 derivative to introduce an Nco I cloning
site. (B) depicts the pSec-oppA(L25M)-Tn3 construct showing the
layout of the promoter, signal peptide, Tn3 and His tag modules
within the construct.
[0073] FIG. 24. Preparation of secreted Tn3 constructs. (A)
represents the SDS-PAGE analysis of crude media and periplasmic
fractions from E. coli secreting Tn3. (B) represents the SDS-PAGE
analysis of the Tn3 scaffold purified from the E. coli media. (C)
represents the HPLC analysis of the Tn3 purified from the E. coli
media. A shift in retention time following reduction by DTT is
consistent with the presence of a disulfide bond.
[0074] FIG. 25. SDS-PAGE of a SYNAGIS.RTM.-Fc bivalent binder.
Presented in this panel is the reducing (lane 1) and non-reducing
(lane 2) SDS-PAGE analysis of the SynBP01 scaffold fused to an Fc
region. These results demonstrate that the fusion is the correct
size and may indicate dimerization.
[0075] FIG. 26. Comparative binding analysis of a SYNAGIS.RTM.-Fc
bivalent binder with SynBP01. Comparative BIAcore analysis of
SynBP01 vs. SynBP01-Fc. Briefly, SYNAGIS.RTM. was immobilized on
the surface of a CM5 sensor chip through amine coupling. SynBP01 or
SynBP01-Fc, at a concentration of 1 .mu.M, was injected at a flow
rate of 75 .mu.L/min. The bivalent SynBP01-Fc construct exhibits a
higher affinity than the single domain binder.
[0076] FIG. 27. Tn3 scaffolds may be conjugated to PEG. (A)
SDS-PAGE analysis of immobilized metal-affinity column purification
of STn3(CTC) (lanes 1-4), and of purified STn3(CTC) after treatment
with maleimide-derivatized PEG (prior to cation-exchange
chromatography) (lane 5). Lane 1: Total cell lysate from
STn3(CTC)-expressing cells; Lane 2: Flow-through from IMAC column;
Lane 3: Wash fraction from IMAC column; Lane 4: STn3(CTC),
unPEGylated; Lane 5: PEGylated STn3(CTC). (B) SDS-PAGE analysis of
PEGylated STn3 (CTC), as purified from SP XL cation-exchange
column. Peak gradient fractions are shown in lanes 1-5.
[0077] FIG. 28. The AB, CD, and EF loops demonstrate loop-length
diversity. The length of AB, CD and EF loops was extracted for each
of 103 Fn3 sequences and this data was used to produce the loop
length frequency distribution shown. For any given loop, the sum of
frequencies for all lengths is equal to 103, the number of
sequences analyzed.
[0078] FIG. 29. Melting temperature determinations for Tn3 at
different pH and ionic strength. The graphs show the thermal
melting curve determinations, as measured by differential scanning
calorimetry, for the Tn3 scaffold at pH 7.0 (in 20 mM sodium
phosphate buffer) (A), at pH 5.0 (in 20 mM sodium acetate
buffer)(B) and in high salt buffer at pH 7.0 (20 mM sodium
phosphate buffer containing 1.0 M salt)(C). The T.sub.m's were
determined to be about 45.degree. C. for Tn3 at pH 7.0, 52.degree.
C. at pH 5.0, and 55.degree. C. at pH 7.0 in the presence of high
ionic strength. Repeat scans (in yellow) for the pH 7.0 samples
show that thermal unfolding is reversible under these conditions.
Thermal unfolding of Tn3 is irreversible at pH 5.0
[0079] FIG. 30A. Design of charge mutants of Tn3. A cartoon
representation of the three dimensional structure of Tn3 is shown
(pdb code: 1 ten). Residues 8-90 of SEQ ID 1 are shown, with the
side chains of all Asp and Glu residues shown in yellow and white.
A panel of 8 mutants were designed in which the Asp and Glu
residues shown in yellow were replaced with Asn or Gln. Residue
numbering is according to SEQ ID 1.
[0080] FIG. 30B. SDS-PAGE analysis of purified recombinant charge
mutants of Tn3. Aliquots of each of the purified proteins was run
on an SDS-PAGE gel. For comparison, the wild type protein was run
in a separate lane of the same gel, and shows a similar migration
rate to the various charge mutants.
[0081] FIG. 31A. Additivity of stabilizing Tn3 mutations.
[0082] A series of Tn3 point mutants were generated in which
individual Asp or Glu residues were replaced with Asn or Gln.
Presented here are the results from a urea denaturation experiment
in which the intrinsic fluorescence measures the unfolding of the
proteins. In this experiment, 5 of 8 charge mutants of the Tn3
scaffold (E33Q, D49N, E52Q, D53N, E86Q) required a higher
concentration of urea to effect unfolding as compared to the wild
type Tn3 scaffold.
[0083] FIG. 31B. Combination of charge mutations leads to additive
improvements in Tn3 stability.
[0084] Point mutations of Asp or Glu residues which enhanced the
stability of wild type Tn3 were combined into double or triple
mutations of Tn3. Each of the combined Tn3 mutants (E33Q/D49N,
D49N/E86Q and E33Q/D49N/E86Q) exhibited greater stability than any
of the corresponding point mutants or wild type Tn3.
[0085] FIG. 32. Melting temperature determinations for wild type
and charge engineered Tn3 scaffolds. The graph illustrates the
thermal melting curve determinations of the Tn3 scaffold variants
at pH 7.0 as measured by differential scanning calorimetry. The
T.sub.m's were determined to be about 45.degree. C. for wild type
Tn3(A), 50.degree. C. for E33Q/D49N Tn3 (B), 52.degree. C. for
D49N/E86Q Tn3 (C) and 52.degree. C. for E33Q/D49N/E86Q Tn3 (D).
6. DETAILED DESCRIPTION
[0086] The protein scaffolds described herein have been designed to
be superior both to antibody-derived fragments and to non-antibody
frameworks. The major advantage of the scaffolds of the invention
over antibody fragments is structural. These scaffolds are derived
from whole, stable, and soluble structural modules found in human
body fluid proteins and from other sources in nature (for example,
but not limited, to thermophilic bacteria). Consequently, they
exhibit better folding and thermostability properties than antibody
fragments, whose creation involves the removal of parts of the
antibody native fold, often exposing amino acid residues that, in
an intact antibody, would be buried in a hydrophobic environment,
such as an interface between variable and constant domains.
Exposure of such hydrophobic residues to solvent increases the
likelihood of aggregation.
[0087] Moreover, the scaffolds of the invention provide the
functional advantages of antibody molecules. In particular, despite
the fact that the scaffold is not an immunoglobulin, its overall
fold is close to that of the variable region of the IgG heavy
chain, making it possible to display its three loops in an
analogous fashion to antibody CDRs in relative orientations.
Because of this structure, the scaffolds of the invention possess
antigen binding properties that are similar in nature and affinity
to those of antibodies. As a result, loop randomization and
shuffling strategies may be employed in vitro that are similar to
the process of affinity maturation of antibodies in vivo.
6.1 The FnIII Structural Motif
[0088] The scaffolds of the present invention are based on the
structure of a fibronectin module of type III (FnIII), a domain
found in mammalian blood and structural proteins. This domain
occurs often in the proteins sequenced to date, including
fibronectins, tenascin, intracellular cytoskeletal proteins,
cytokine receptors and prokaryotic enzymes (Bork and Doolittle,
Proc. Natl. Acad. Sci. USA 89:8990, 1992; Bork et al., Nature
Biotech. 15:553, 1997; Meinke et al., J. Bacteriol. 175:1910, 1993;
Watanabe et al., J. Biol. Chem. 265:15659, 1990). Although the
domain appears many times in nature, the amino acid sequences are
quite divergent. In particular, these scaffolds include, the third
FnIII domain of tenascin C (also known as the "Tn3" domain). The
overall fold of this domain is closely related to that of the
smallest functional antibody fragment, the variable region of the
heavy chain, which comprises the entire antigen recognition unit in
camel and llama IgG.
[0089] In addition, the Tn3 domain possesses exposed loop sequences
tolerant of randomization, facilitating the generation of diverse
pools of protein scaffolds capable of binding specific targets with
high affinity.
[0090] These protein scaffolds may be utilized for the purpose of
designing proteins which are capable of binding to virtually any
compound (for example, any protein) of interest. In particular, the
molecules based on the Tn3 structural motif described herein may be
used as scaffolds which are subjected to directed evolution
designed to randomize one or more of the loops which are analogous
to the complementarity-determining regions (CDRs) of an antibody
variable region. Such a directed evolution approach results in the
production of antibody-like molecules with high affinities for
antigens of interest. In addition, the scaffolds described herein
may be used to display defined exposed loops (for example, loops
previously randomized and selected on the basis of antigen binding)
in order to direct the evolution of molecules that bind to such
introduced loops. A selection of this type may be carried out to
identify recognition molecules for any individual CDR-like loop or,
alternatively, for the recognition of two or all three CDR-like
loops combined into a non-linear epitope binding moiety.
[0091] A set of three loops of Tn3 corresponding to the
antigen-binding loops of the IgG heavy chain run between amino acid
residues 23-31 (BC), 50-56 (DE), and 75-84 (FG). The length of the
BC, DE, and FG loops, 9, 6, and 10 residues, respectively, fall
within the narrow range of the corresponding antigen-recognition
loops found in antibody heavy chains, that is, 7-10, 4-8, and 4-28
residue lengths, respectively. Alternatively, in another
embodiment, the BC, DE, and FG loops may run between amino acid
residues 23 to 31, 51 to 56, and 75 to 84 respectively.
Accordingly, once randomized and selected for high antigen
affinity, these loops may make contacts with antigens equivalent to
the contacts of the corresponding loops in antibodies. The AB, CD,
and EF loops of the Tn3 domain also share this property and hence,
also are available for randomization and selection for high
affinity for antigens. This process may be accomplished in parallel
or in series with the randomization of the BC, DE, and FG
loops.
[0092] In a specific embodiment, one or more loop regions of the
scaffold based on the Tn3 domain of human tenascin C comprise amino
acid residues: [0093] I. From 12 to 17 inclusive in an AB loop;
[0094] II. From 23 to 31 inclusive in a BC loop; [0095] III. From
39 to 45 inclusive in a CD loop; [0096] IV. From 50 to 56 inclusive
in a DE loop; [0097] V. From 60 to 66 inclusive in an EF loop; and
[0098] VI. From 75 to 84 inclusive in an FG loop. In another
specific embodiment, scaffolds of the invention comprise at least
one, at least two, at least three, at least four, at least five, or
at least six loops wherein a loop comprises an amino acid sequence
of SEQ ID NOs: 201, 202, 203, 204, 205, or 206. In one embodiment,
scaffolds of the invention comprise an AB loop having an amino acid
sequence of SEQ ID NO:201. In another embodiment, scaffolds of the
invention comprise a BC loop having an amino acid sequence of SEQ
ID NO:202. In another embodiment, scaffolds of the invention
comprise a CD loop having an amino acid sequence of SEQ ID NO:203.
In another embodiment, scaffolds of the invention comprise a DE
loop having an amino acid sequence of SEQ ID NO:204. In another
embodiment, scaffolds of the invention comprise an EF loop having
an amino acid sequence of SEQ ID NO:205. In another embodiment,
scaffolds of the invention comprise a FG loop having an amino acid
sequence of SEQ ID NO:206. In a specific embodiment, scaffolds of
the invention comprise an AB loop having an amino acid sequence of
SEQ ID NO:201, a BC loop having an amino acid sequence of SEQ ID
NO:202, a CD loop having an amino acid sequence of SEQ ID NO:203, a
DE loop having an amino acid sequence of SEQ ID NO:204, an EF loop
having an amino acid sequence of SEQ ID NO:205, and an FG loop
having an amino acid sequence of SEQ ID NO:206.
[0099] In another specific embodiment, one or more loop regions of
the scaffold based on the Tn3 domain of human tenascin C comprise
amino acid residues: [0100] I. From 11 to 17 inclusive in an AB
loop; [0101] II. From 23 to 31 inclusive in a BC loop; [0102] III.
From 39 to 45 inclusive in a CD loop; [0103] IV. From 51 to 56
inclusive in a DE loop; [0104] V. From 60 to 67 inclusive in an EF
loop; and [0105] VI. From 75 to 84 inclusive in an FG loop.
[0106] In another specific embodiment, scaffolds of the invention
comprise at least one, at least two, at least three, at least four,
at least five, or at least six loops wherein a loop comprises an
amino acid sequence of SEQ ID NOs: 207, 202, 203, 208, 209, or 206.
In one embodiment, scaffolds of the invention comprise an AB loop
having an amino acid sequence of SEQ ID NO:207. In another
embodiment, scaffolds of the invention comprise a BC loop having an
amino acid sequence of SEQ ID NO:202. In another embodiment,
scaffolds of the invention comprise a CD loop having an amino acid
sequence of SEQ ID NO:203. In another embodiment, scaffolds of the
invention comprise a DE loop having an amino acid sequence of SEQ
ID NO:208. In another embodiment, scaffolds of the invention
comprise an EF loop having an amino acid sequence of SEQ ID NO:209.
In another embodiment, scaffolds of the invention comprise a FG
loop having an amino acid sequence of SEQ ID NO:206. In a specific
embodiment, scaffolds of the invention comprise an AB loop having
an amino acid sequence of SEQ ID NO:207, a BC loop having an amino
acid sequence of SEQ ID NO:202, a CD loop having an amino acid
sequence of SEQ ID NO:203, a DE loop having an amino acid sequence
of SEQ ID NO:208, an EF loop having an amino acid sequence of SEQ
ID NO:209, and an FG loop having an amino acid sequence of SEQ ID
NO:206.
[0107] In other embodiments, scaffolds of the invention comprise
loop regions that are variants of the cognate loop regions in any
of SEQ ID NOs:1-32 or 68-88.
[0108] The invention provides recombinant, non-naturally occurring
polypeptide scaffolds comprising, a plurality of beta strand
domains linked to a plurality of loop region sequences derived from
a naturally occurring protein sequence, wherein one or more of said
loop region sequences vary by deletion, substitution or addition by
at least one amino acid from the corresponding loop sequences in
the naturally occurring protein sequence, and wherein the beta
strand domains of the polypeptide scaffold have at least 50%
homology to the corresponding domain sequences of a naturally
occurring protein sequence. For example, such amino acid sequences
may be, but are not limited to, any of SEQ ID NOs: 1-32, 60-88, and
210. In another specific embodiment, the scaffold of the invention
comprises the sequence of the Tn3 domain of human tenascin C. In
another embodiment, the scaffold of the invention comprises a
sequence having at least 50% homology to the Tn3 domain of human
tenascin C. In further embodiments, said homology to the Tn3 domain
is at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99%, or more. In other embodiments, the naturally occurring
sequence is a protein sequence corresponding to an additional Tn3
structural motif from human tenascin C. In other embodiments, the
naturally occurring sequence is a protein sequence corresponding to
a Tn3 structural motif from another tenascin protein, or
alternatively, a tenascin protein from another organism (such as,
but not limited to, murine, porcine, bovine, or equine
tenascins).
[0109] Although Tn3 represents a scaffold for the generation of
antibody mimics, other molecules may be substituted for Tn3 in the
molecules described herein. These include, without limitation,
related Tn3 structural motifs from animals and prokaryotes. In
addition, Tn3 structural motifs from other proteins may also be
used. Modules from different organisms and parent proteins may be
most appropriate for different applications; for example, in
designing a scaffold stable at a low pH, it may be most desirable
to generate that protein from organism that optimally grows at a
low pH (such as, but not limited to Sulfolobus tokodaii). In
another embodiment, related Tn3 structural motifs may be identified
and utilized from hyperthermophillic bacteria such as, but not
limited to Archaeoglobus fulgidus and Staphylothermus marinus, each
of which exhibit optimal growth at greater than 70.degree. C. In
other embodiments, the naturally occurring sequence corresponds to
a predicted Tn3 structural motif from a thermophilic organism, for
example, but not limited to Archaeoglobus fulgidus, Staphylothermus
marinus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, and
Sulfolobus tokodaii. In yet another embodiment, the scaffolds of
the invention have a protein sequence having at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95% or at least 99% homology
to any of the sequences from a sequence corresponding to a Tn3
structural motif or a predicted Tn3 structural motif from a
thermophillic organism as described above. In some embodiments, the
Tn3 structural motifs from thermophillic organisms may be selected
from the amino acid sequences of SEQ ID NOs.:2-4, and 68-88.
[0110] The invention also provides scaffolds of the invention with
a plurality of beta strands of more than seven. In one embodiment,
scaffolds of the invention comprise a plurality at least 7, at
least 8, at least 9, at least 10, at least 11 or more beta
strands.
[0111] The invention also provides scaffolds of the invention with
a plurality of loop regions of more than six. In one embodiment,
scaffolds of the invention comprise a plurality at least 7, at
least 8, at least 9, at least 10, at least 11 or more loop
regions.
[0112] In one embodiment, scaffolds of the invention comprise at
least seven beta strands, wherein said beta strands are designated
N-terminus to C-terminus A, B, C, D, E, F, and G strands. In
another embodiment, the scaffolds of the invention comprise at
least seven beta strands, each strand separated by a loop region
wherein the loop regions are designated N-terminus to C-terminus,
AB, BC, CD, DE, EF, and FG loops. In alternative embodiment, the
scaffolds of the invention comprise less than seven beta strands,
each strand separated by a loop region. In an alternate embodiment,
the scaffolds of the invention comprise less than seven beta
strands, each strand separated by a loop region.
[0113] In another specific embodiment, scaffolds of the invention
comprise at least one, at least two, at least three, at least four,
at least five, at least six, or at least seven beta strands,
wherein said beta strands comprise amino acid sequences selected
from SEQ ID NOs:228-234. In another specific embodiment, scaffolds
of the invention comprise at least seven beta strands, designated
N-terminus to C-Terminus A-G, wherein said A strand comprises the
sequence of SEQ ID NO:228. In another specific embodiment,
scaffolds of the invention comprise at least seven beta strands,
designated N-terminus to C-Terminus A-G, wherein said B strand
comprises the sequence of SEQ ID NO:229. In another specific
embodiment, scaffolds of the invention comprise at least seven beta
strands, designated N-terminus to C-Terminus A-G, wherein said C
strand comprises the sequence of SEQ ID NO:230. In another specific
embodiment, scaffolds of the invention comprise at least seven beta
strands, designated N-terminus to C-Terminus A-G, wherein said D
strand comprises the sequence of SEQ ID NO:231. In another specific
embodiment, scaffolds of the invention comprise at least seven beta
strands, designated N-terminus to C-Terminus A-G, wherein said E
strand comprises the sequence of SEQ ID NO:232. In another specific
embodiment, scaffolds of the invention comprise at least seven beta
strands, designated N-terminus to C-Terminus A-G, wherein said F
strand comprises the sequence of SEQ ID NO:233. In another specific
embodiment, scaffolds of the invention comprise at least seven beta
strands, designated N-terminus to C-Terminus A-G, wherein said G
strand comprises the sequence of SEQ ID NO:234. In another specific
embodiment, scaffolds of the invention comprise an A strand having
the sequence of SEQ ID NO:228, a B strand having the sequence of
SEQ ID NO:229, a C strand having the sequence of SEQ ID NO:230, a D
strand having the sequence of SEQ ID NO:231, an E strand having the
sequence of SEQ ID NO:232, an F strand having the sequence of SEQ
ID NO:233 and a G strand having the sequence of SEQ ID NO:234.
[0114] In another specific embodiment, scaffolds of the invention
comprise at least one, at least two, at least three, at least four,
at least five, at least six, or at least seven beta strands,
wherein said beta strands comprise amino acid sequences selected
from SEQ ID NOs:235, 229, 230, 236, 232, 237, and 234. In another
specific embodiment, scaffolds of the invention comprise at least
seven beta strands, designated N-terminus to C-Terminus A-G,
wherein said A strand comprises the sequence of SEQ ID NO:235. In
another specific embodiment, scaffolds of the invention comprise at
least seven beta strands, designated N-terminus to C-Terminus A-G,
wherein said B strand comprises the sequence of SEQ ID NO:229. In
another specific embodiment, scaffolds of the invention comprise at
least seven beta strands, designated N-terminus to C-Terminus A-G,
wherein said C strand comprises the sequence of SEQ ID NO:230. In
another specific embodiment, scaffolds of the invention comprise at
least seven beta strands, designated N-terminus to C-Terminus A-G,
wherein said D strand comprises the sequence of SEQ ID NO:236. In
another specific embodiment, scaffolds of the invention comprise at
least seven beta strands, designated N-terminus to C-Terminus A-G,
wherein said E strand comprises the sequence of SEQ ID NO:232. In
another specific embodiment, scaffolds of the invention comprise at
least seven beta strands, designated N-terminus to C-Terminus A-G,
wherein said F strand comprises the sequence of SEQ ID NO:237. In
another specific embodiment, scaffolds of the invention comprise at
least seven beta strands, designated N-terminus to C-Terminus A-G,
wherein said G strand comprises the sequence of SEQ ID NO:234. In
another specific embodiment, scaffolds of the invention comprise an
A strand having the sequence of SEQ ID NO:235, a B strand having
the sequence of SEQ ID NO:229, a C strand having the sequence of
SEQ ID NO:230, a D strand having the sequence of SEQ ID NO:236, an
E strand having the sequence of SEQ ID NO:232, an F strand having
the sequence of SEQ ID NO:237 and a G strand having the sequence of
SEQ ID NO:234.
[0115] In another embodiment, scaffolds of the invention comprises
beta strand sequences having at least 50% homology to the cognate
beta strands of any of SEQ ID NOs:1-32 or 68-99. In further
embodiments, said homology is at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 99%, or more.
[0116] The loops connecting the various strands of the protein
scaffold may be randomized for length and/or sequence diversity. In
one embodiment, the scaffolds of the invention have at least one
loop is randomized for length and/or sequence diversity. In another
embodiment, the scaffolds of the invention have at least one loop
is kept constant while at least one additional loop is randomized
for length and/or sequence diversity. In another embodiment, the
libraries of the invention have scaffolds where at least one of
loops AB, CD, and EF are kept constant while at least one of loops
BC, DE, and FG are randomized for length or sequence diversity.
[0117] In a specific embodiment, the scaffolds of the invention
comprise a BC loop which is randomized with the following consensus
sequence: S-X-a-X-b-X-X-X-G, wherein X represents any amino acid,
wherein (a) represents proline or alanine and wherein (b)
represents alanine or glycine.
[0118] In another specific embodiment the scaffolds of the
invention comprise a BC loop which is randomized with the following
consensus sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X represents
any amino acid and wherein (d) represents proline, glutamate or
lysine, wherein (e) represents asparagine or glycine, and wherein
(f) represents serine or glycine.
[0119] In another embodiment, the scaffolds of the invention have a
BC loop which comprises 11 amino acids having a consensus sequence
of S-P-c-X-X-X-X-X-X-T-G, wherein X represents any amino acid and
wherein (c) represents proline, serine or glycine.
[0120] In a specific embodiment, the scaffolds of the invention
comprise an FG loop which is randomized with the following
consensus sequence: X-a-X-X-G-X-X-X-S, wherein X represents any
amino acid and wherein (a) represents asparagine, threonine or
lysine.
[0121] In another specific embodiment, the scaffolds of the
invention comprise an FG loop which is randomized with the
following consensus sequence: X-a-X-X-X-X-b-N-P-A, wherein X
represents any amino acid, wherein (a) represents asparagine,
threonine or lysine and wherein (b) represents serine or
glycine.
[0122] In another specific embodiment, the scaffolds of the
invention comprise a an FG loop which is randomized with the
following consensus sequence: X-a-X-X-G-X-X-S-N-P-A, wherein X
represents any amino acid, and wherein (a) represents asparagine,
threonine or lysine.
[0123] In a specific embodiment, the libraries of the invention
comprise scaffolds with a DE loop, comprising 6 residues, which is
randomized with the following consensus sequence: X-X-X-X-X-X,
wherein X represents any amino acid.
[0124] In a specific embodiment, the scaffolds of the invention
comprise an AB loop, comprising 7 residues, which is randomized
with the following consensus sequence: K-X-X-X-X-X-a, wherein X
represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, and wherein (a) represents serine, threonine,
alanine, or glycine.
[0125] In a specific embodiment, the scaffolds of the invention
comprise an AB loop, comprising 9 residues, which is randomized
with the following consensus sequence: K-X-X-X-X-X-X-X-a, wherein X
represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, and wherein (a) represents serine, threonine,
alanine, or glycine.
[0126] In a specific embodiment, the scaffolds of the invention
comprise a CD loop, comprising 7, 8, or 9 residues, wherein each
residue in the CD loop is randomized and wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine.
[0127] In a specific embodiment, the scaffolds of the invention
comprise an EF loop comprising 8 residues, which is randomized with
the following consensus sequence: X-b-L-X-P-X-c-X, wherein X
represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, wherein (b) represents asparagine, lysine,
arginine, aspartic acid, glutamic acid, or glycine, and wherein (c)
represents isoleucine, threonine, serine, valine, alanine, or
glycine.
[0128] In some embodiments, the scaffolds of the invention may
comprise about 75 to about 500, about 75 to about 200, about 75 to
about 100, about 75 to about 250, or about 75 to about 150 amino
acids.
6.2 Disulfide-Engineered Scaffold-Based Proteins
[0129] In an effort to increase the stability of scaffolds of the
invention, a bioinformatics approach was utilized to identify
candidate positions suitable for engineering of a disulfide bond.
However, disulfide design by manual inspection of protein
structures to identify candidate residue pairs in close proximity
is often unproductive due to the strict geometric constraints
required by that type of bond (see Dombkowski, Bioinformatics Vol.
19 No. 14, 2003 1852-1853). Thus, the present invention provides
scaffolds having disulfide bonds engineered at positions that
exhibit enhanced stability as measured by thermal tolerance,
resistance to chaotropic denaturation and protease treatment.
[0130] In one embodiment, the scaffolds of the invention comprise
at least one, at least two, at least three, at least four, or at
least five non-naturally occurring disulfide bonds. In one
embodiment, the scaffolds of the invention comprise a least one
non-naturally occurring disulfide bond, wherein said at least one
non-naturally occurring disulfide bond stabilizes the scaffold. In
another embodiment, the scaffolds of the invention comprise at
least one non-naturally occurring disulfide bond located between
two beta strands. For example, said at least one non-naturally
occurring disulfide bond may form a link between the A strand and B
strand, or between the D strand and E strand, or between the F
strand and G strand, or between the C strand and F strand. In
another embodiment, said at least one non-naturally occurring
disulfide bond forms a first bond between the F strand and the G
strand, and a second link between the C strand and F strand. In
another embodiment, the scaffolds of the invention comprise at
least one non-naturally occurring disulfide bond located between
two loop regions. In another embodiment, the scaffolds of the
invention comprise at least one non-naturally occurring disulfide
bond located between a loop region and a beta strand. In another
embodiment, scaffolds of the invention comprise at least one
non-naturally occurring disulfide bond that is located within the
same beta strand. In another embodiment, scaffolds of the invention
comprise at least one non-naturally occurring disulfide bond that
is located within the same loop region. In another embodiment,
scaffolds of the invention comprise at least one non-naturally
occurring disulfide bond, wherein the bond is located between two
distinct scaffolds.
[0131] In another embodiment, the scaffolds of the invention
comprise a disulfide bond that forms a multimeric scaffold (the
term "multimeric" is defined as at least two or more scaffolds in
association) of at least 2, at least 3, at least 4 or more
scaffolds.
[0132] The increase in stability contributed by the engineering of
disulfide bonds can be readily measured by techniques well known in
the art, such as thermal (T.sub.m) and chaotropic denaturation
(such as urea, or guanidine), protease treatment (such as
thermolysin) or another art accepted stability parameter. A
comprehensive review of techniques used to measure protein
stability can be found, for example in "Current Protocols in
Molecular Biology" and "Current Protocols in Protein Science" by
John Wiley and Sons. 2007.
[0133] In one embodiment the disulfide containing scaffolds of the
invention exhibit an increase in stability of at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% or more compared
to the same scaffold prior to disulfide engineering, as measured by
thermal tolerance, resistance to chaotropic denaturation, protease
treatment or another stability parameter well-known in the art.
[0134] The stability of a protein may be measured by the level of
fluorescence exhibited by the protein under varying conditions.
There is a positive correlation between the relative unfoldedness
of a protein and a change in the internal fluorescence the protein
exhibits under stress. Suitable protein stability assays to measure
thermal characteristics include Differential Scanning Calorimetry
(DSC) and Circular Dichroism (CD). When the protein demonstrates a
sizable shift in parameters measured by DSC or CD, it correlates to
an unfolded structure, the temperature at which this shift is made
is termed the melting temperature or (T.sub.m). In one embodiment,
the disulfide engineered scaffolds of the invention exhibit an
increased melting temperature (T.sub.m) of at least greater than
45.degree. C., at least greater than 50.degree. C., at least
greater than 55.degree. C., at least greater than 60.degree. C., at
least greater than 65.degree. C., at least greater than 70.degree.
C., at least greater than 71.degree. C., at least greater than
72.degree. C., at least greater than 73.degree. C., at least
greater than 74.degree. C., at least greater than 75.degree. C., at
least greater than 76.degree. C., at least greater than 77.degree.
C., at least greater than 78.degree. C., at least greater than
79.degree. C., at least greater than 80.degree. C., at least
greater than 81.degree. C., at least greater than 82.degree. C., at
least greater than 83.degree. C., at least greater than 84.degree.
C., at least greater than 85.degree. C., at least greater than
85.degree. C., at least greater than 86.degree. C., at least
greater than 87.degree. C., at least greater than 88.degree. C., at
least greater than 89.degree. C., at least greater than 90.degree.
C., at least greater than 91.degree. C., at least greater than
92.degree. C., at least greater than 93.degree. C., at least
greater than 94.degree. C., at least greater than 94.degree. C., at
least greater than, at least greater than 95.degree. C., at least
greater than 96.degree. C., at least greater than 97.degree. C. or
at least greater than 98.degree. C., or at least greater than
100.degree. C., or at least greater than 105.degree., or at least
greater than 110.degree., or at least greater than 120.degree. than
the melting temperature (T.sub.m) exhibited by the same scaffold
prior to engineering under the same experimental conditions.
[0135] In another embodiment, the disulfide engineered scaffolds of
the invention exhibit an increased melting temperature (T.sub.m) of
at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, or at least
95% or more compared to the same scaffold prior to disulfide
engineering under the same experimental conditions.
[0136] Another assay for protein stability involves exposing a
protein to a chaotropic agent, such as urea or guanidine (for
example, guanidine-HCl or guanidine isothiocynate) which acts to
destabilize interactions within the protein. Upon exposing the
protein to increasing levels of urea or guanidine, the relative
internal fluorescence is measured to asses a value in which 50% of
the protein molecules are unfolded. This value is termed the
C.sub.m value and represents a benchmark value for protein
stability. The higher the C.sub.m value, the more stable the
protein. In one embodiment, the disulfide engineered scaffolds of
the invention exhibit an increased C.sub.m at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% or more compared
to the same scaffold prior to disulfide engineering as measured in
a urea denaturation experiment under similar conditions.
[0137] In another embodiment, the disulfide engineered scaffolds of
the invention exhibit an increased C.sub.m at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% or more compared
to the same scaffold prior to disulfide engineering as measured in
a guanidine denaturation experiment under similar conditions.
[0138] Another assay used to assay protein stability is a protease
resistance assay. In this assay, a relative level of protein
stability is correlated with the resistance to protease degradation
over time. The more resistant to protease treatment, the more
stable the protein is. In one embodiment, the disulfide engineered
scaffolds of the invention exhibit increased stability by at least
5%, at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, or at least 95% or
more compared to the same scaffold prior to disulfide engineering
as measured in a protease resistance experiment under similar
conditions.
[0139] In some instances it may be advantageous to utilize a
scaffold of the invention with decreased stability, for example but
not limited to, a scaffold conjugated to a cytotoxin, or a
radionuclide. Such scaffolds may require faster clearance rates
related to non-specific toxicity. In one embodiment, the scaffolds
of the invention comprise a disulfide bond that de-stabilizes the
scaffold as compared to the scaffold prior to engineering. In one
embodiment the disulfide containing scaffolds of the invention
exhibit a decrease in stability of at least 5%, at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% or more compared to the
same scaffold prior to disulfide engineering, as measured by
thermal tolerance, resistance to chaotropic denaturation, protease
treatment or another stability parameter well-known in the art
under similar experimental conditions.
6.3 Scaffold Kinetics
[0140] The invention provides scaffolds that specifically bind a
target (for example, a protein). In some embodiments, the target
may be, for example, but not limited to, a cell-surface antigen, a
soluble antigen, an immobilized antigen, an immunosilent antigen,
an intracellular antigen, an intranuclear antigen, a self antigen,
a non-self antigen, a cancer antigen, a bacterial antigen, or a
viral antigen.
[0141] In some embodiments, scaffolds of the invention specifically
bind a target with specific kinetics. In some embodiments,
scaffolds of the invention may have an association rate constant or
k.sub.on rate (scaffold(Sc)+antigen (Ag)(k.sub.on.fwdarw.Sc--Ag) of
at least 10.sup.5 M.sup.-1 s.sup.-1, at least
1.5.times.10.sup.5M.sup.-1 s.sup.-1, at least 2.times.10.sup.5
M.sup.-1 s.sup.-1, at least 2.5.times.10.sup.5 M.sup.-1 s.sup.-1,
at least 5.times.10.sup.5 M.sup.-1 s.sup.-1, at least 10.sup.6
M.sup.-1 s.sup.-1, at least 5.times.10.sup.6 M.sup.-1 s.sup.-1, at
least 10.sup.7 M.sup.-1 s.sup.-1, at least 5.times.10.sup.7M.sup.-1
s.sup.-1, or at least 10.sup.8 M.sup.-1 s.sup.-1, or a k.sub.on
rate of about 10.sup.5 to about 10.sup.8M.sup.-1 S.sup.-1, a
k.sub.on rate of about 1.5.times.10.sup.5 M.sup.-1 s.sup.-1 to
about 1.times.10.sup.7 M.sup.-1 s.sup.-1, a k.sub.on rate of about
2.times.10.sup.5 to about 1.times.10.sup.6M.sup.-1 s.sup.-1, or a
k.sub.on rate of about 4.5.times.10.sup.5 to about 5.times.10.sup.7
M.sup.-1 s.sup.-1 as determined by a BIAcore.RTM. assay or by other
assays known in the art.
[0142] In some embodiments, scaffolds of the invention may have a
k.sub.off rate (Scaffold (Sc)+antigen (Ag k.sub.offSc--Ag) of less
than 10.sup.-3 s.sup.-1, less than 5.times.10.sup.-3 s.sup.-1, less
than 10.sup.-4 s.sup.-1, less than 2.times.10.sup.-4 s.sup.-1, less
than 5.times.10.sup.-4 s.sup.-1, less than 10.sup.-5 s.sup.-1, less
than 5.times.10.sup.-5 S.sup.-1, less than 10.sup.-6 S.sup.-1, less
than 5.times.10.sup.-6 s.sup.-1, less than 10.sup.-7 s.sup.-1, less
than 5.times.10.sup.-7 S.sup.-1, less than 10.sup.-8 S.sup.-1, less
than 5.times.10.sup.-8 s.sup.-1, less than 10.sup.-9 s.sup.-1, less
than 5.times.10.sup.-9 S.sup.-1, or less than 10.sup.-10 s.sup.-1,
or 10.sup.-3-10.sup.-10 s.sup.-1, 10.sup.-4-10.sup.-8 s.sup.-1, or
10.sup.-5-10.sup.-8 s.sup.-1 as determined by a BIAcore.RTM. assay
or by other assays known in the art.
[0143] In some embodiments, scaffolds of the invention may have an
affinity constant or K.sub.a (k.sub.on/k.sub.off) of at least
10.sup.2 M.sup.-1, at least 5.times.10.sup.2 M.sup.-1, at least
10.sup.3 M.sup.-1, at least 5.times.10.sup.3 M.sup.-1, at least
10.sup.4 M.sup.-1, at least 5.times.10.sup.4 M.sup.-1, at least
10.sup.5 M.sup.-1, at least 5.times.10.sup.5 M.sup.-1, at least
10.sup.6M.sup.-1, at least 5.times.10.sup.6 M.sup.-1, at least
10.sup.7 M.sup.-1, at least 5.times.10.sup.7 M.sup.-1, at least
10.sup.8M.sup.-1, at least 5.times.10.sup.8 M.sup.-1, at least
10.sup.9 M.sup.-1, at least 5.times.10.sup.9 M.sup.-1, at least
10.sup.10 M.sup.-1, at least 5.times.10.sup.10 M.sup.-1, at least
10.sup.11 M.sup.-1, at least 5.times.10.sup.11 M.sup.-1, at least
10.sup.12M.sup.-1, at least 5.times.10.sup.12 M.sup.-1, at least
10.sup.13 M.sup.-1, at least 5.times.10.sup.13 M.sup.-1, at least
10.sup.14 M.sup.-1, at least 5.times.10.sup.14 M.sup.-1, at least
10.sup.15 M.sup.-1, or at least 5.times.10.sup.5 M.sup.-1, or
10.sup.2-5.times.10.sup.5 M.sup.-1, 10.sup.4-1.times.10.sup.10
M.sup.-1, or 10.sup.5-1.times.10.sup.8 M.sup.-1. Scaffolds of the
invention may have a K.sub.a of at most 10.sup.11M.sup.-1, at most
5.times.10.sup.11 M.sup.-1, at most 10.sup.12 M.sup.-1, at most
5.times.10.sup.12 M.sup.-1, at most 10.sup.13 M.sup.-1, at most
5.times.10.sup.13 M.sup.-1, at most 10.sup.14M.sup.-1, or at most
5.times.10.sup.14 M.sup.-1 as determined by a BIAcore.RTM. assay or
by other assays known in the art.
[0144] In some embodiments, scaffolds of the invention may have a
dissociation constant or K.sub.d (k.sub.off/k.sub.on) of less than
10.sup.-5 M, less than 5.times.10.sup.-5M, less than 10.sup.-6M,
less than 5.times.10.sup.-6M, less than 10.sup.-7 M, less than
5.times.10.sup.-7, less than 10.sup.-8 M, less than
5.times.10.sup.-8 M, less than 10.sup.-9 M, less than
5.times.10.sup.-9 M, less than 10.sup.-10 M, less than
5.times.10.sup.-10 M, less than 10.sup.-11M, less than
5.times.10.sup.-11 M, less than 10.sup.-12 M, less than
5.times.10.sup.-12 M, less than 10.sup.-13 M, less than
5.times.10.sup.-13 M, less than 10.sup.-14 M, less than
5.times.10.sup.-14M, less than 10.sup.15 M, or less than
5.times.10.sup.-15 M as determined by a BIAcore Assay.RTM. or by
other assays known in the art.
6.4 Directed Evolution of Scaffold-Based Binding Proteins
[0145] The scaffolds described herein may be used in any technique
for evolving new or improved target binding proteins. In one
particular example, the target is immobilized on a solid support,
such as a column resin or microtiter plate well, and the target
contacted with a library of candidate scaffold-based binding
proteins. Such a library may consist of clones constructed from the
Tn3 motif based scaffold through randomization of the sequence
and/or the length of the CDR-like loops. In one embodiment, the
library may be a phage, phagemid, virus, bacterial or yeast display
or a ribosome display library. If desired, this library may be an
RNA-protein fusion library generated, for example, by the
techniques described in Szostak et al., U.S. Pat. No. 6,258,558 B1,
U.S. Pat. No. 6,261,804 B1; U.S. Pat. No. 5,643,768 and U.S. Pat.
No. 5,658,754. Alternatively, it may be a DNA-protein library (for
example, as described in Lohse, DNA-Protein Fusions and Uses
Thereof, U.S. Ser. No. 60/110,549, filed Dec. 2, 1998, now
abandoned, and U.S. Ser. No. 09/453,190, filed Dec. 2, 1999).
[0146] In this regard, bacteriophage (phage) display is one well
known technique which allows one to screen large oligopeptide
libraries to identify member(s) of those libraries which are
capable of specifically binding to a target. Phage display is a
technique by which variant polypeptides are displayed as fusion
proteins to the coat protein on the surface of bacteriophage
particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386).
The utility of phage display lies in the fact that large libraries
of selectively randomized protein variants (or randomly cloned
cDNAs) can be rapidly and efficiently sorted for those sequences
that bind to a target molecule with high affinity. Display of
peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA,
87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D.
et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been
used for screening millions of polypeptides or oligopeptides for
ones with specific binding properties (Smith, G. P. (1991) Current
Opin. Biotechnol., 2:668). Sorting phage libraries of random
mutants requires a strategy for constructing and propagating, a
large number of variants, a procedure for affinity purification
using the target receptor, and a means of evaluating the results of
binding enrichments (see for example, U.S. Pat. Nos. 5,223,409,
5,403,484, 5,571,689, and 5,663,143).
[0147] Although most phage display methods have used filamentous
phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No.
5,627,024), T4 phage display systems (Ren et al., Gene, 215: 439
(1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998);
Jiang et al., Infection & Immunity, 65(11): 4770-4777 (1997);
Ren et al., Gene, 195(2):303-311 (1997); Ren, Protein Sci., 5: 1833
(1996); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage
display systems (Smith and Scott, Methods in Enzymology, 217:
228-257 (1993); U.S. Pat. No. 5,766,905) are also known.
[0148] Many other improvements and variations of the basic phage
display concept have now been developed. These improvements enhance
the ability of display systems to screen peptide libraries for
binding to selected target molecules and to display functional
proteins with the potential of screening these proteins for desired
properties. Combinatorial reaction devices for phage display
reactions have been developed (WO 98/14277) and phage display
libraries have been used to analyze and control bimolecular
interactions (WO 98/20169; WO 98/20159) and properties of
constrained helical peptides (WO 98/20036). WO 97/35196 describes a
method of isolating an affinity ligand in which a phage display
library is contacted with one solution in which the ligand will
bind to a target molecule and a second solution in which the
affinity ligand will not bind to the target molecule, to
selectively isolate binding ligands. WO 97/46251 describes a method
of biopanning a random phage display library with an affinity
purified antibody and then isolating binding phage, followed by a
micropanning process using microplate wells to isolate high
affinity binding phage. The use of Staphlylococcus aureus protein A
as an affinity tag has also been reported (Li et al. (1998) Mol.
Biotech., 9:187). WO 97/47314 describes the use of substrate
subtraction libraries to distinguish enzyme specificities using a
combinatorial library which may be a phage display library. A
method for selecting enzymes suitable for use in detergents using
phage display is described in WO 97/09446. Additional methods of
selecting specific binding proteins are described in U.S. Pat. Nos.
5,498,538, 5,432,018, and WO 98/15833.
[0149] Methods of generating peptide libraries and screening these
libraries are also disclosed in U.S. Pat. Nos. 5,723,286,
5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018,
5,698,426, 5,763,192, and 5,723,323.
[0150] For the present libraries of the invention, a bioinformatics
approach was employed to determine the loop length and diversity
preferences of naturally occurring Tn3 structural motifs (see
Example 2). During this analysis, the preferences for loop length
and sequence diversity were employed to develop a "restricted
randomization" approach. In this restricted randomization, the
relative loop length and sequence preferences were incorporated
into the development of a library strategy. For example, it was
determined, that one loop length preference for the BC loop was 9
residues. Upon further analysis of 9 residue containing BC loops it
was determined whether there was a preference for a particular
amino acid, or group of amino acids at that position or if the
position was completely random. Integrating the loop length and
sequence diversity analysis into library development resulted in a
restricted randomization (i.e. certain positions within the
randomized loop were limited in which amino acid could reside in
that position). Examples of the restricted randomization approach
are described in the Examples (see Example 2).
[0151] The invention also provides libraries (hereinafter referred
to as "libraries of the invention") comprising scaffolds of the
invention described herein. In one embodiment, the libraries of the
invention comprise non-naturally occurring polypeptide scaffolds
comprising, a plurality of beta strand domains linked to a
plurality of loop region sequences derived from a naturally
occurring protein sequence, wherein one or more of said loop region
sequences vary by deletion, substitution or addition by at least
one amino acid from the corresponding loop sequences in the
naturally occurring protein sequence, and wherein the beta strand
domains of the polypeptide scaffold have at least 50% homology to
the corresponding domain sequences of a naturally occurring protein
sequence. In some embodiments the naturally occurring sequence is
the protein sequence corresponding to human tenascin Tn3. In other
embodiments, the naturally occurring sequence is a protein sequence
corresponding to an additional Tn3 structural motif from human
tenascin C. In other embodiments, the naturally occurring sequence
is a protein sequence corresponding to a Tn3 structural motif from
another tenascin protein, or alternatively, a tenascin protein from
another organism (such as, but not limited to, murine, porcine,
bovine, or equine tenascins). In yet another embodiment, the
naturally occurring sequence is a protein sequence corresponding to
a Tn3 structural motif from any organism. In other embodiments, the
naturally occurring sequence corresponds to a predicted Tn3
structural motif from a thermophilic organism, for example, but not
limited to Archaeoglobus fulgidus, Staphylothermus marinus,
Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus
tokodaii. In yet another embodiment, the scaffolds of the invention
have a protein sequence having at least 30%, at least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95% or at least 99% homology to any of the
sequences corresponding to a Tn3 structural motif or a predicted
Tn3 structural motif as described above.
[0152] The library is incubated with the immobilized target, the
support is washed to remove non-specific binders, and the tightest
binders are eluted under very stringent conditions and subjected to
PCR to recover the sequence information or to create a new library
of binders which may be used to repeat the selection process, with
or without further mutagenesis of the sequence. A number of rounds
of selection may be performed until binders of sufficient affinity
for the antigen are obtained.
[0153] In another embodiment, the libraries of the invention
comprise scaffolds described herein. In one embodiment, libraries
of the invention comprise scaffolds further comprising at least
seven beta strands, wherein said beta strands are designated
N-terminus to C-terminus A, B, C, D, E, F, and G strands. In
another embodiment, the libraries of the invention comprise
scaffolds which further comprise at least seven beta strands, each
strand separated by a loop region wherein the loop regions are
designated N-terminus to C-terminus, AB, BC, CD, DE, EF, and FG
loops. In another embodiment, libraries of the invention comprise
scaffolds further comprising at least seven beta strands,
designated N-terminus to C-terminus A, B, C, D, E, F and G strands
wherein each strand is connected by a loop region wherein the loop
regions are designated N-terminus to C-terminus, AB, BC, CD, DE,
EF, and FG.
[0154] In a specific embodiment, the libraries of the invention
comprise scaffolds further comprising one or more loop regions of
the scaffold based on the Tn3 domain of human tenascin C comprising
amino acid residues: [0155] I. from 12 to 17 inclusive in an AB
loop; [0156] II. from 23 to 31 inclusive in an BC loop; [0157] III.
from 39 to 45 inclusive in an CD loop; [0158] IV. from 50 to 56
inclusive in an DE loop; [0159] V. from 60 to 66 inclusive in an EF
loop; and [0160] VI. from 75 to 84 inclusive in an FG loop.
[0161] In another specific embodiment, libraries of the invention
comprise scaffolds further comprising one or more loop regions of
the scaffold based on the Tn3 domain of human tenascin C comprising
amino acid residues: [0162] I. From 11 to 17 inclusive in an AB
loop; [0163] II. From 23 to 31 inclusive in a BC loop; [0164] III.
From 39 to 45 inclusive in a CD loop; [0165] IV. From 51 to 56
inclusive in a DE loop; [0166] V. From 60 to 67 inclusive in an EF
loop; and [0167] VI. From 75 to 84 inclusive in an FG loop.
[0168] The invention also provides libraries comprising scaffolds
comprising loop sequence diversity. In one embodiment, the
libraries of the invention comprise scaffolds with at least one
loop which contains at least one position that is randomized. In
another embodiment, libraries of the invention comprise scaffolds
with at least one loop which comprises at least one position that
is randomized while further comprising at least one position that
is held constant. In another embodiment, the libraries of the
invention comprise scaffolds with a loop which comprises at least
one position that is subjected to a restricted randomization. In
another embodiment, the libraries of the invention comprise
scaffolds with at least one loop which comprises at least one
position that is subjected to a restricted randomization and
further comprises at least one position that is held constant. In
another embodiment, the libraries of the invention comprise
scaffolds with at least one loop which comprises at least one
position that is subjected to a restricted randomization and
further comprises at least one position that is randomized and at
least one position that is held constant.
[0169] The loops connecting the various strands of the protein
scaffold may be randomized for length and/or sequence diversity. In
one embodiment, the libraries of the invention have scaffolds with
at least one loop is randomized for length and/or sequence
diversity. In another embodiment, the libraries of the invention
have scaffolds where at least one loop is kept constant while at
least one additional loop is randomized for length and/or sequence
diversity. In another embodiment, the libraries of the invention
have scaffolds where at least one, at least two, or all three of
loops AB, CD, and EF are kept constant while at least one, at least
two, or all three of loops BC, DE, and FG are randomized for length
or sequence diversity. In another embodiment, the libraries of the
invention have scaffolds wherein at least one, at least two, or at
least all three of loops AB, CD, and EF are randomized while at
least one, at least two, or all three of loops BC, DE, and FG are
randomized for length or sequence diversity.
[0170] In a specific embodiment, the libraries of the invention
comprise scaffolds with a BC loop which is randomized with the
following consensus sequence: S-X-a-X-b-X-X-X-G, wherein X
represents any amino acid, wherein (a) represents proline or
alanine and wherein (b) represents alanine or glycine.
[0171] In another specific embodiment the scaffolds of the
invention comprise a BC loop which is randomized with the following
consensus sequence: A-d-P-X-X-X-e-f-X-I-X-G, wherein X represents
any amino acid and wherein (d) represents proline, glutamate or
lysine, wherein (e) represents asparagine or glycine, and wherein
(f) represents serine or glycine.
[0172] In another embodiment, the libraries of the invention have a
BC loop which comprises 11 amino acids having a consensus sequence
of S-P-c-X-X-X-X-X-X-T-G, wherein X represents any amino acid and
wherein (c) represents proline, serine or glycine.
[0173] In a specific embodiment, the libraries of the invention
comprise scaffolds with an FG loop which is randomized with the
following consensus sequence: X-a-X-X-G-X-X-X-S, wherein X
represents any amino acid and wherein (a) represents asparagine,
threonine or lysine.
[0174] In another specific embodiment, the libraries of the
invention comprise scaffolds with an FG loop which is randomized
with the following consensus sequence: X-a-X-X-X-X-b-N-P-A, wherein
X represents any amino acid, wherein (a) represents asparagine,
threonine or lysine and wherein (b) represents serine or
glycine.
[0175] In another specific embodiment, the libraries of the
invention comprise a scaffold with an FG loop which is randomized
with the following consensus sequence: X-a-X-X-G-X-X-S-N-P-A,
wherein X represents any amino acid, and wherein (a) represents
asparagine, threonine or lysine.
[0176] In a specific embodiment, the libraries of the invention
comprise scaffolds with an AB loop, comprising 7 residues, which is
randomized with the following consensus sequence: K-X-X-X-X-X-a,
wherein X represents asparagine, aspartic acid, histidine,
tyrosine, isoleucine, valine, leucine, phenylalanine, threonine,
alanine, proline, or serine, and wherein (a) represents serine,
threonine, alanine, or glycine.
[0177] In a specific embodiment, the libraries of the invention
comprise scaffolds with an AB loop, comprising 9 residues, which is
randomized with the following consensus sequence:
K-X-X-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid,
histidine, tyrosine, isoleucine, valine, leucine, phenylalanine,
threonine, alanine, proline, or serine, and wherein (a) represents
serine, threonine, alanine, or glycine.
[0178] In a specific embodiment, the libraries of the invention
comprise scaffolds with a CD loop, comprising 7, 8, or 9 residues,
wherein each residue in the CD loop is randomized and wherein X
represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine.
[0179] In a specific embodiment, the libraries of the invention
comprise scaffolds with an EF loop comprising 8 residues, which is
randomized with the following consensus sequence: X-b-L-X-P-X-c-X,
wherein X represents asparagine, aspartic acid, histidine,
tyrosine, isoleucine, valine, leucine, phenylalanine, threonine,
alanine, proline, or serine, and wherein (b) represents asparagine,
lysine, serine, arginine, aspartic acid, glutamic acid, or glycine,
and wherein (c) represents isoleucine, threonine, serine, valine,
alanine, or glycine.
[0180] A further embodiment of the invention is a collection of
isolated nucleic acid molecules encoding a library comprising the
scaffolds of the invention and as described above.
[0181] A further practical consideration in the design of these Tn3
libraries was to identify an alternative to the "NNK" (N=A, G, T,
C; K=G, T) mixed codon scheme typically used in degenerate
oligonucleotides to code for any amino acid. Although the "NNK"
mixture gives 32 different codons which code for all 20 amino
acids, they are not encoded equally (Table 11). For instance, 3/32
codons in the "NNK" scheme code for Leu (CTG, CTT, TTG), but only
1/32 codes for Asp (GAT). In addition, the "NNK" mixture encodes
one stop codon (TAG) and a Cys codon (TGT), neither of which is
desirable when generating naive libraries. In considering an
alternative scheme, we took note of the fact that synthetic
antibody libraries have been described which encode CDR sequences
composed of a small subset of amino acids. Antibody libraries with
CDR's composed of just 4 amino acids (Tyr, Ala, Asp, Ser), or even
a binary pair (Tyr, Ser) have been shown to yield specific high
affinity mAbs to protein antigens (Fellouse et al, Proc Natl Acad
Sci, 2004, 101:12467-72, Fellouse et al. J. Mol. Biol., 2005,
348:1153-62). Similarly, a library of 10Fn3 scaffold proteins with
randomized loop sequences comprising just Tyr and Ser also yielded
specific binders to a protein target (Koide et al. Proc Natl Acad,
Sci, 2007, 104:66-32-7). Although libraries containing highly
restricted sets of amino acids are able to produce specific binding
proteins, it is likely that the diversity of binders that are
obtained from a library will be limited. We therefore designed an
alternate "NHT" mixed codon scheme for introducing diversity into a
Tn3 library (H=A, T, C). "NHT" mixes code for a reasonable subset
of the 20 amino acids, but avoid the disadvantages described with
"NNK" mixed codons (Table 12). This scheme generates 12 codons that
code for 12/20 amino acids, that is, each codon codes for a unique
amino acid. Moreover, there are no stop or Cys codons. Accordingly,
in some embodiments, scaffolds of the invention comprise codons
encoded by the NHT codon scheme. In other embodiments, scaffolds of
the invention comprise codons encoded by the NNK mixed codon
scheme.
[0182] In other embodiments, the scaffolds of the invention may be
subjected to affinity maturation. In this art-accepted process, a
specific binding protein is subject to a scheme that selects for
increased affinity for a specific target (see Wu et al. Proc Natl
Aca Sci USA. May 1998 26; 95(11):6037-42). The resultant scaffolds
of the invention may exhibit binding characteristics at least as
high as compared to the scaffolds prior to affinity maturation.
[0183] In other embodiments, the scaffolds of the invention may be
subjected to "loop grafting" analogous to CDR grafting for
antibodies. In this art-accepted process, one or more CDRs from an
antibody are "grafted" onto an acceptor antibody (or, in this
example, a scaffold of the invention (see Ewert et al. Methods:2004
October; 34(2):184-99). In another embodiment, at least one loop
from another scaffold may be grafted onto a scaffold of the
invention.
[0184] The invention also provides methods of identifying the amino
acid sequence of a protein scaffold capable of binding to target so
as to form a scaffold:target complex. In one embodiment, the method
comprises the steps of: a) providing a polypeptide display library
of the invention; b) contacting the polypeptide display library of
(a) with an immobilized or separable target; c) separating the
scaffold:target complexes from the free scaffolds; d) causing the
replication of the separated scaffolds of (c) so as to result in a
new polypeptide display library distinguished from that in (a) by
having a lowered diversity and by being enriched in displayed
scaffolds capable of binding the target; e) optionally repeating
steps (b), (c), and (d) with the new library of (d); and
determining the nucleic acid sequence of the region encoding the
displayed scaffold of a species from (d) and hence deducing the
peptide sequence capable of binding to the target.
[0185] In another embodiment, the scaffolds of the invention may be
further randomized after identification from a library screen. In
one embodiment, methods of the invention comprise further
randomizing at least one, at least two, at least three, at least
four, at least five or at least six loops of a scaffold identified
from a library using a method described herein. In another
embodiment, the further randomized scaffold is subjected to a
subsequent method of identifying a scaffold capable of binding a
target, said method comprising (a) contacting said further
randomized scaffold with an immobilized or separable target, (b)
separating the further randomized scaffold:target complexes from
the free scaffolds, (c) causing the replication of the separated
scaffolds of (b), optionally repeating steps (a)-(c), and (d)
determining the nucleic acid sequence of the region encoding said
further randomized scaffold and hence, deducing the peptide
sequence capable of binding to the target. In a further embodiment,
the further randomized scaffolds comprise at least one, at least
two, at least three, at least four, at least five, or at least six
randomized loops which were previously randomized in the first
library. In an alternate further embodiment, the further randomized
scaffolds comprise at least one, at least two, at least three, at
least four, at least five, or at least six randomized loops which
were not previously randomized in the first library.
[0186] In another embodiment, the scaffolds of the invention may be
further randomized after identification from a library screen. In
one embodiment, methods of the invention comprise further
randomizing at least one, at least two, at least three, at least
four, at least five, at least six or at least seven strands of a
scaffold identified from a library using a method described herein.
In another embodiment, the further randomized scaffold is subjected
to a subsequent method of identifying a scaffold capable of binding
a target, said method comprising (a) contacting said further
randomized scaffold with an immobilized or separable target, (b)
separating the further randomized scaffold:target complexes from
the free scaffolds, (c) causing the replication of the separated
scaffolds of (b), optionally repeating steps (a)-(c), and (d)
determining the nucleic acid sequence of the region encoding said
further randomized scaffold and hence, deducing the peptide
sequence capable of binding to the target. In a further embodiment,
the further randomized scaffolds comprise at least one, at least
two, at least three, at least four, at least five, at least six, or
at least seven randomized strands which were previously randomized
in the first library. In an alternate further embodiment, the
further randomized scaffolds comprise at least one, at least two,
at least three, at least four, at least five, at least six, or at
least seven randomized strands which were not previously randomized
in the first library.
[0187] In another embodiment, the scaffolds of the invention may be
further randomized after identification from a library screen. In
one embodiment, methods of the invention comprise further
randomizing at least one, at least two, at least three, at least
four, at least five, or at least six and at least one, at least
two, at least three, at least four, at least five, at least six, or
at least seven strands of a scaffold identified from a library
using a method described herein. In another embodiment, the further
randomized scaffold is subjected to a subsequent method of
identifying a scaffold capable of binding a target, said method
comprising (a) contacting said further randomized scaffold with an
immobilized or separable target, (b) separating the further
randomized scaffold:target complexes from the free scaffolds, (c)
causing the replication of the separated scaffolds of (b),
optionally repeating steps (a)-(c), and (d) determining the nucleic
acid sequence of the region encoding said further randomized
scaffold and hence, deducing the peptide sequence capable of
binding to the target. In a further embodiment, the further
randomized scaffolds comprise at least one, at least two, at least
three, at least four, at least five, or at least six randomized
loops and at least one, at least two, at least three, at least
four, at least five, at least six, or at least seven strands which
were previously randomized in the first library. In an alternate
further embodiment, the further randomized scaffolds comprise at
least one, at least two, at least three, at least four, at least
five, or at least six randomized loops and at least one, at least
two, at least three, at least four, at least five, at least six, or
at least seven strands which were not previously randomized in the
first library.
[0188] In another embodiment, one method of obtaining a scaffold of
the invention involves a first randomized loop selected from the
group consisting of BC, DE, and FG loops and a second loop not
randomized in said library selected from the group consisting of
AB, CD, and EF loops. In yet another embodiment, another method of
obtaining a scaffold of the invention involves a first randomized
loop selected from the group consisting of AB, CD, EF loops and a
second loop not randomized in said library selected from the group
consisting of BC, DE, and FG loops.
[0189] The invention also provides a method obtaining at least two
scaffolds that bind to at least one or more targets. This method
allows for the screening of agents that act cooperatively to elicit
a particular response. It may be advantageous to use such a screen
when an agonistic activity requiring the cooperation of more than
one scaffold is required (for example, but not limited to, agonism
of a receptor tyrosine kinase). This method allows for the
screening of cooperative agents without the reformatting of the
library to form multimeric complexes. In one embodiment, the method
of the invention comprises contacting a target ligand with a
library of the invention under conditions that allow a
scaffold:target ligand complex to form, engaging said scaffolds
with a crosslinking agent (defined as an agent that brings
together, in close proximity, at least two identical or distinct
scaffolds) wherein the crosslinking of the scaffolds elicits a
detectable response and obtaining from the complex, said scaffolds
that bind the target. In a further embodiment, the crosslinking
agent is a scaffold specific antibody, or fragment thereof, an
epitope tag specific antibody of a fragment thereof, a dimerization
domain, such as Fc region, a leucine zipper motif, a chemical
crosslinker, or another dimerization domain known in the art.
[0190] The invention also provides methods of detecting a compound
utilizing the scaffolds of the invention. Based on the binding
specificities of the scaffolds obtained by library screening, it is
possible to use such scaffolds in assays to detect the specific
target in a sample, such as for diagnostic methods. In one
embodiment, the method of detecting a compound comprises contacting
said compound in a sample with a scaffold of the invention, under
conditions that allow a compound:scaffold complex to form and
detecting said scaffold, thereby detecting said compound in a
sample. In further embodiments, the scaffold is labeled (i.e.
radiolabel, fluorescent, enzyme-linked or colorimetric label) to
facilitate the detection of said compound.
[0191] The invention also provides methods of capturing a compound
utilizing the scaffolds of the invention. Based on the binding
specificities of the scaffolds obtained by library screening, it is
possible to use such scaffolds in assays to capture the specific
target in a sample, such as for purification methods. In one
embodiment, the method of capturing a compound in a sample
comprises contacting said compound in a sample with a scaffold of
the invention under conditions that allow the formation of a
compound:scaffold complex and removing said complex from the
sample, thereby capturing said compound in said sample. In further
embodiments, the scaffold is immobilized to facilitate the removing
of the compound:scaffold complex.
[0192] In some embodiments, scaffolds isolated from libraries of
the invention comprise at least one, at least two, at least four,
at least five, at least six, or more randomized loop regions. In
some embodiments, isolated scaffold loop sequences may be swapped
from a donor scaffold to any loop in a receiver scaffold (for
example, an AB loop sequence from a donor scaffold may be
transferred to an y loop region in a receiver scaffold). In
specific embodiments, an isolated loop sequences may be transferred
to the cognate loop in the receiving scaffold (for example, an AB
loop sequence from a donor scaffold may be transferred to a
receiver scaffold in the AB loop position). In some embodiments,
isolated loop sequences may be "mix and matched" randomly with
various receiver scaffolds.
[0193] In other embodiments, isolated scaffolds sequences may be
identified by the loop sequence. For example, a library is used to
pan against a particular target and an collection of specific
binders are isolated. The randomized loop sequences may be
characterized as specific sequences independently of the scaffold
background (i.e., The scaffold that binds target X wherein said
scaffold comprises an AB loop sequence of SEQ ID NO:x). In
alternative embodiments, where a scaffold exhibits two loop
sequences that bind target X, the loop sequences may be
characterized as binding target X in the absence of the scaffold
sequence. In other words, it is contemplated that scaffolds
isolated from a library that bind a particular target may be
expressed as the variable loop sequences that bind that target
independent of the scaffold backbone. This process would be
analogous to the concept of CDRs in variable regions of
antibodies.
[0194] In some embodiments, the invention provides scaffolds
comprising sequences that specifically bind SYNAGIS.RTM.. In such
embodiments, scaffolds of the invention that specifically bind
SYNAGIS.RTM. may comprise an BC loop sequence selected from SEQ ID
NOs:100, 102, 105, 107, and 109. In other embodiments, scaffolds of
the invention that specifically bind SYNAGIS.RTM. may comprise an
FG loop sequence selected from SEQ ID NOs:101, 103, 104, 106, 108,
or 110. In further embodiments, scaffolds of the invention that
specifically bind SYNAGIS.RTM. may comprise at least one BC loop
sequence selected from SEQ ID NOs:100, 102, 105, 107, and 109; and
may comprise at least one FG loop sequence selected from SEQ ID
NOs:101, 103, 104, 106, 108, or 110. In other embodiments, the
invention may also comprise scaffolds which compete for binding
with scaffolds that specifically bind SYNAGIS.RTM., said
SYNAGIS.RTM. binders comprising one BC loop sequence selected from
SEQ ID NOs:100, 102, 105, 107, and 109; and comprising an FG loop
sequence selected from SEQ ID NOs:101, 103, 104, 106, 108, or 110.
Competition assays may be performed as presented herein in Examples
11 and/or 14, or by other assays known in the art.
[0195] In some embodiments, the invention provides scaffolds
comprising sequences that specifically bind lysozyme. In such
embodiments, scaffolds of the invention that specifically bind
lysozyme may comprise at least one BC loop sequence selected from
SED ID NOs:111, 114, 102, 120, and 124. In other embodiments,
scaffolds of the invention that specifically bind lysozyme may
comprise at least one FG loop sequence selected from SEQ ID NOs:
112, 113, 115, 116, 117, 118, 119, 121, 122, or 125. In further
embodiments, scaffolds of the invention that specifically bind
lysozyme may comprise at least one BC loop sequence selected from
SED ID NOs:111, 114, 102, 120, and 124; and may comprise at least
one FG loop sequence selected from SEQ ID NOs: 112, 113, 115, 116,
117, 118, 119, 121, 122, or 125. In other embodiments, the
invention may also comprise scaffolds which compete for binding
with scaffolds that bind lysozyme, said lysozyme binders comprising
at least one BC loop sequence selected from SED ID NOs:111, 114,
102, 120, or 124; and at least one FG loop sequence selected from
SEQ ID NOs: 112, 113, 115, 116, 117, 118, 119, 121, 122, or 125.
Competition assays may be performed as presented herein in Examples
11 and/or 14, or by other assays known in the art.
6.5 Multimeric Scaffolds
[0196] In addition to scaffold monomers, any of the scaffold
constructs described herein may be generated as dimers or multimers
of scaffolds as a means to increase the valency and thus the
avidity of antigen binding. Also, any of the scaffold constructs
described herein may be generated as dimers or multimers of
scaffolds as a means to increase the specificity of antigen binding
(for example, scaffolds may be generated that bind distinct
antigens). Such multimers (multimeric scaffolds or also known
herein as multivalent scaffolds) may be generated through covalent
binding between individual scaffold modules, for example, by the
inclusion of an amino acid linker. In other methods, the multimeric
scaffolds may be assembled through use of dimerization domains
known in the art. In particular examples, covalently bonded
scaffolds may be generated by constructing fusion genes that encode
the monomeric scaffolds or, alternatively, by engineering codons
for cysteine residues into monomer sequences and allowing disulfide
bond formation to occur between the expression products.
[0197] Non-covalently bonded multimeric scaffolds may also be
generated by a variety of techniques. These include the
introduction, into monomer sequences, of codons corresponding to
positively and/or negatively charged residues and allowing
interactions between these residues in the expression products (and
therefore between the monomers) to occur. This approach may be
simplified by taking advantage of charged residues naturally
present in a monomer subunit. Another means for generating
non-covalently bonded scaffolds is to introduce, into the monomer
scaffold gene (for example, at the amino- or carboxy-termini), the
coding sequences for proteins or protein domains known to interact.
Such proteins or protein domains include coil-coil motifs, leucine
zipper motifs, and any of the numerous protein subunits (or
fragments thereof) known to direct formation of dimers or higher
order multimers.
[0198] In some embodiments, multimeric scaffolds of the invention
comprise at least one scaffold fused to any domain (or fragment) of
an antibody. In some embodiments, at least one scaffold is fused to
an antibody variable domain, a CH1 domain, a Ckappa domain, a
Clambda domain, a CH2, or a CH3 domain. In other embodiments, at
least one scaffold is fused to the CH2 domain of an antibody Fc. In
such embodiments, the resulting protein, when expressed will be
bivalent for a particular target through the dimerization of the
CH2 and CH3 regions of the antibody Fc fragment. In further
embodiments, the scaffold of the invention replaces the antibody
variable region connected to the Fc fragment. In alternative
embodiments, the scaffold of the invention does not replace the
antibody variable region connected to the CH1-Fc fragment, Ckappa
or Clambda domains.
[0199] In further embodiments, multimeric scaffolds are constructed
by fusing scaffolds to the CH1 and the Ckappa or Clamdba regions of
an antibody. In some embodiments, the scaffolds of the invention
replace the antibody variable regions fused to the CH1 and the
Ckappa or Clambda regions of an antibody. In further embodiments,
scaffolds of the invention may be fused to the C-terminus of the
light chain or heavy chain of an antibody. In other embodiments,
scaffolds of the invention may be fused to the N-terminus of the
light chain or heavy chain of an antibody.
[0200] In some embodiments, multimeric scaffolds of the invention
comprise scaffolds that are specific for the same epitope. In other
embodiments, multimeric scaffolds of the invention comprise
scaffolds that are specific for different epitopes otherwise known
as an epitope binding domain. Multimeric scaffolds of the invention
may be assembled and utilized as shown in "Multispecific epitope
binding proteins and uses thereof" U.S. application Ser. No.
12/182,975, filed Jul. 30, 2008 which is hereby incorporated by
reference in its entirety for all purposes. Such epitope binding
domains may be selected from an antibody, an antibody fragment, a
diabody, an scFv, a Fab, a Fv, or a binding peptide.
[0201] In other embodiments, the epitope binding domain will be
specific for the same target as that of the scaffold of the
invention.
[0202] In another embodiment, the epitope binding domain will be
specific for a different target as that of the scaffold of the
invention.
[0203] Choosing a suitable linker for a specific case where two or
more scaffolds are to be connected may depend on a variety of
parameters including, e.g. the nature of the monomer domains,
and/or the stability of the peptide linker towards proteolysis and
oxidation.
[0204] The linker polypeptide may predominantly include amino acid
residues selected from the group consisting of Gly, Ser, Ala and
Thr. For example, the peptide linker may contain at least 75%
(calculated on the basis of the total number of residues present in
the peptide linker), such as at least 80%, e.g. at least 85% or at
least 90% of amino acid residues selected from the group consisting
of Gly, Ser, Ala and Thr. The peptide linker may also consist of
Gly, Ser, Ala and/or Thr residues only. The linker polypeptide
should have a length, which is adequate to link two or more monomer
domains of the invention or two or more multimeric scaffolds of the
invention in such a way that they assume the correct conformation
relative to one another so that they retain the desired
activity.
[0205] A suitable length for this purpose is a length of at least
one and typically fewer than about 50 amino acid residues, such as
2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid
residues, 8-12 amino acid residues or 11 residues. Similarly, the
polypeptide encoding a linker can range in size, e.g., from about 2
to about 15 amino acids, from about 3 to about 15, from about 4 to
about 12, about 10, about 8, or about 6 amino acids. In methods and
compositions involving nucleic acids, such as DNA, RNA, or
combinations of both, the polynucleotide containing the linker
sequence can be, e.g., between about 6 nucleotides and about 45
nucleotides, between about 9 nucleotides and about 45 nucleotides,
between about 12 nucleotides and about 36 nucleotides, about 30
nucleotides, about 24 nucleotides, or about 18 nucleotides.
Likewise, the amino acid residues selected for inclusion in the
linker polypeptide should exhibit properties that do not interfere
significantly with the activity or function of the polypeptide
multimer. Thus, the peptide linker should on the whole not exhibit
a charge which would be inconsistent with the activity or function
of the polypeptide multimer, or interfere with internal folding, or
form bonds or other interactions with amino acid residues in one or
more of the monomer domains which would seriously impede the
binding of the polypeptide multimer to specific targets.
[0206] The peptide linker may also be selected from a library where
the amino acid residues in the peptide linker are randomized for a
specific set of monomer domains in a particular polypeptide
multimer. A flexible linker could be used to find suitable
combinations of monomer domains, which is then optimized using this
random library of variable linkers to obtain linkers with optimal
length and geometry. The optimal linkers may contain the minimal
number of amino acid residues of the right type that participate in
the binding to the target and restrict the movement of the monomer
domains relative to each other in the polypeptide multimer when not
bound to specific targets.
[0207] The use of naturally occurring as well as artificial peptide
linkers to connect polypeptides into novel linked fusion
polypeptides is well known in the literature (Hallewell et al.
(1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995),
Protein Eng. 8, 725-731; Robinson & Sauer (1996), Biochemistry
35, 109-116; Khandekar et al. (1997), J. Biol. Chem. 272,
32190-32197; Fares et al. (1998), Endocrinology 139, 2459-2464;
Smallshaw et al. (1999), Protein Eng. 12, 623-630; U.S. Pat. No.
5,856,456).
[0208] As mentioned above, it is generally preferred that the
peptide linker possess at least some flexibility. Accordingly, in
some embodiments, the peptide linker contains 1-25 glycine
residues, 5-20 glycine residues, 5-15 glycine residues or 8-12
glycine residues. The peptide linker will typically contain at
least 50% glycine residues, such as at least 75% glycine residues.
In some embodiments of the invention, the peptide linker comprises
glycine residues only. In specific embodiments, linker sequences
may comprise a sequence of (G-G-G-G-S).sub.x where x is a positive
integer. In another specific embodiment, linker sequences may
comprise a sequence of (G-A).sub.x where x is a positive integer.
In another specific embodiment, linker sequences may comprise a
sequence of (G-G-G-T-P-T).sub.x where x is a positive integer.
[0209] In some cases it may be desirable or necessary to provide
some rigidity into the peptide linker. This may be accomplished by
including proline residues in the amino acid sequence of the
peptide linker. Thus, in another embodiment of the invention, the
peptide linker may comprise at least one proline residue in the
amino acid sequence of the peptide linker. For example, the peptide
linker has an amino acid sequence, wherein at least 25%, such as at
least 50%, e.g. at least 75%, of the amino acid residues are
proline residues. In one particular embodiment of the invention,
the peptide linker comprises proline residues only.
[0210] In some embodiments of the invention, the peptide linker is
modified in such a way that an amino acid residue comprising an
attachment group for a non-polypeptide moiety is introduced.
Examples of such amino acid residues may be a cysteine residue (to
which the non-polypeptide moiety is then subsequently attached) or
the amino acid sequence may include an in vivo N-glycosylation site
(thereby attaching a sugar moiety (in vivo) to the peptide linker).
An additional option is to genetically incorporate non-natural
amino acids using evolved tRNAs and tRNA synthetases (see, e.g.,
U.S. patent application Publication Ser. No. 2003/0082575) into the
monomer domains or linkers. For example, insertion of keto-tyrosine
allows for site-specific coupling to expressed monomer domains or
multimers.
[0211] Sometimes, the amino acid sequences of all peptide linkers
present in the polypeptide multimer will be identical.
Alternatively, the amino acid sequences of all peptide linkers
present in the polypeptide multimer may be different.
6.6 Fusions
[0212] The scaffolds described herein may be fused to other protein
domains. For example, these scaffolds may be integrated with the
human immune response by fusing the constant region of an IgG (Fc)
with a scaffold, through the N or C-terminus The Fc fusion molecule
activates the complement component of the immune response and
increases the therapeutic value of the protein scaffold. Similarly,
a fusion between a scaffold and a complement protein, such as Clq,
may be used to target cells, and a fusion between scaffold and a
toxin may be used to specifically destroy cells that carry a
particular antigen.
[0213] Additionally, various publications describe methods for
obtaining physiologically active molecules whose half-lives are
modified either by introducing an FcRn-binding polypeptide into the
molecules (WO 97/43316; U.S. Pat. No. 5,869,046; U.S. Pat. No.
5,747,035; WO 96/32478; WO 91/14438) or by fusing the molecules
with antibodies whose FcRn-binding affinities are preserved but
affinities for other Fc receptors have been greatly reduced (WO
99/43713) or fusing with FcRn binding domains of antibodies (WO
00/09560; U.S. Pat. No. 4,703,039). Specific techniques and methods
of increasing half-life of physiologically active molecules can
also be found in U.S. Pat. No. 7,083,784 granted Aug. 1, 2006
entitled "Antibodies with Increased Half-lives" which is hereby
incorporated by reference for all purposes. Specifically, it is
contemplated that the scaffolds of the invention can be fused to an
Fc region from an IgG, wherein said Fc region comprises amino acid
residue mutations (as numbered by the EU index in Kabat):
M252Y/S254T/T256E or H433K/N434F/Y436H.
[0214] In addition, the scaffolds of the invention may be fused
with molecules that increases or extends in vivo or serum half
life. In some embodiments, the scaffolds of the invention associate
with albumin, such as human serum albumin (HSA), polyethylene
glycol (PEG), polysaccharides, immunoglobulin molecules (IgG),
complement, hemoglobin, a binding peptide, lipoproteins and other
factors to increase its half-life in the bloodstream and/or its
tissue penetration. Any of these fusions may be generated by
standard techniques, for example, by expression of the fusion
protein from a recombinant fusion gene constructed using publicly
available gene sequences.
[0215] Also, the scaffolds of the invention may bind or associate
with molecules that increases or extends in vivo or serum half
life. In some embodiments, the scaffolds of the invention associate
with albumin, polyethylene glycol (PEG), polysaccharides,
immunoglobulin molecules or immunoglobulin molecules having Fc
mutations that increase serum half life, complement, hemoglobin,
lipoproteins and other factors to increase serum half life. Any of
these fusions may be generated by standard techniques, for example,
by expression of the fusion protein from a recombinant fusion gene
constructed using publicly available gene sequences.
[0216] The term "polyethylene glycol" or "PEG" means a polyethylene
glycol compound or a derivative thereof, with or without coupling
agents, coupling or activating moieties (e.g., with thiol,
triflate, tresylate, azirdine, oxirane, N-hydroxysuccinimide or a
maleimide moiety). The term "PEG" is intended to indicate
polyethylene glycol of a molecular weight between 500 and 150,000
Da, including analogues thereof, wherein for instance the terminal
OH-group has been replaced by a methoxy group (referred to as
mPEG).
[0217] In one embodiment, the scaffolds are derivatized with
polyethylene glycol (PEG). PEG is a linear, water-soluble polymer
of ethylene oxide repeating units with two terminal hydroxyl
groups. PEGs are classified by their molecular weights which
typically range from about 500 daltons to about 40,000 daltons. In
a presently preferred embodiment, the PEGs employed have molecular
weights ranging from 5,000 daltons to about 20,000 daltons. PEGs
coupled to the scaffolds of the invention can be either branched or
unbranched. (See, for example, Monfardini, C. et al. 1995
Bioconjugate Chem 6:62-69). PEGs are commercially available from
Nektar Inc., Sigma Chemical Co. and other companies. Such PEGs
include, but are not limited to, monomethoxypolyethylene glycol
(MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S),
monomethoxypolyethylene glycol-succinimidyl succinate
(MePEG-S-NHS), monomethoxypolyethylene glycol-amine
(MePEG-NH.sub.2), monomethoxypolyethylene glycol-tresylate
(MePEG-TRES), and monomethoxypolyethylene
glycol-imidazolyl-carbonyl (MePEG-IM).
[0218] Briefly, in one embodiment, the hydrophilic polymer which is
employed, for example, PEG, is capped at one end by an unreactive
group such as a methoxy or ethoxy group. Thereafter, the polymer is
activated at the other end by reaction with a suitable activating
agent, such as cyanuric halides (for example, cyanuric chloride,
bromide or fluoride), diimadozle, an anhydride reagent (for
example, a dihalosuccinic anhydride, such as dibromosuccinic
anhydride), acyl azide, p-diazoiumbenzyl ether,
3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The
activated polymer is then reacted with a polypeptide as described
herein to produce a polypeptide derivatized with a polymer.
Alternatively, a functional group in the scaffolds of the invention
can be activated for reaction with the polymer, or the two groups
can be joined in a concerted coupling reaction using known coupling
methods. It will be readily appreciated that the polypeptides of
the invention can be derivatized with PEG using a myriad of other
reaction schemes known to and used by those of skill in the
art.
[0219] In some embodiments, scaffolds of the invention are
engineered to provide reactive groups for conjugation. In such
scaffolds, the N-terminus and/or C-terminus may also serve to
provide reactive groups for conjugation. In other embodiments, the
N-terminus may be conjugated to one moiety (such as, but not
limited to PEG) while the C-terminus is conjugated to another
moiety (such as, but not limited to biotin), or vice versa.
[0220] The term "in vivo half-life" is used in its normal meaning,
i.e., the time at which 50% of the biological activity of the
polypeptide is still present in the body/target organ, or the time
at which the activity of the polypeptide is 50% of its initial
value. As an alternative to determining functional in vivo
half-life, "serum half-life" may be determined, i.e., the time at
which 50% of the polypeptide molecules circulate in the plasma or
bloodstream prior to being cleared. Determination of
serum-half-life is often more simple than determining functional
half-life and the magnitude of serum-half-life is usually a good
indication of the magnitude of functional in vivo half-life.
Alternative terms to serum half-life include plasma half-life,
circulating half-life, circulatory half-life, serum clearance,
plasma clearance, and clearance half-life. The functionality to be
retained is normally selected from procoagulant, proteolytic,
co-factor binding, receptor binding activity, or other type of
biological activity associated with the particular protein.
[0221] The term "increased" with respect to the functional in vivo
half-life or plasma half-life is used to indicate that the relevant
half-life of the polypeptide is statistically significantly
increased relative to that of a reference molecule (for example an
unmodified polypeptide), as determined under comparable conditions.
For instance the relevant half-life may be increased by at least
about 25%, such as by at least about 50%, e.g., by at least about
100%, at least about 150%, at least about 200%, at least about
250%, or at least about 500% compared to an unmodified reference
molecule. In other embodiments, the half-life may be increased by
about at least 1 fold, at least 2 fold, at least 3 fold, at least 4
fold, at least 5 fold, at least 10 fold, at least 20 fold, or at
least 50 fold as compared to an unmodified reference molecule.
6.7 Randomization Embodiments
[0222] In one aspect, the invention provides randomized scaffolds.
In another embodiment, the invention also provides multimeric
randomized scaffolds. In another embodiment, the invention also
provides disulfide engineered randomized scaffolds. In yet another
embodiment, the invention provides libraries comprising randomized
scaffolds. The randomization scheme of the scaffolds of the
invention and the display libraries comprising said scaffolds,
collectively referred to in this section as "the present invention"
is provided below.
[0223] In one embodiment, scaffolds of the invention comprise at
least 1, at least 2, at least 3, at least 4, at least 5, or at
least 6 randomized loops. In another embodiment, the present
invention comprise at least one randomized loop wherein, at least
the AB, or at least the BC, or at least the CD, or at least the DE,
or at least the EF, or at least the FG loop is randomized.
[0224] In one embodiment, the present invention comprise one
randomized loop. For example, the present invention comprise a
randomized AB loop. In another embodiment, the present invention
comprise a randomized BC loop. In another embodiment, the present
invention comprise a randomized CD loop. In another embodiment, the
present invention comprise a randomized DE loop. In another
embodiment, the present invention comprise a randomized EF loop. In
another embodiment, the present invention comprise a randomized FG
loop.
[0225] In one embodiment, the present invention comprise two
randomized loops. For example, the present invention comprise
randomized AB and BC loops. In another embodiment, the present
invention comprise randomized AB and CD loops. In another
embodiment, the present invention comprise randomized AB and DE
loops. In another embodiment, the present invention comprise
randomized AB and EF loops. In another embodiment, the present
invention comprise randomized AB and FG loops. In another
embodiment, the present invention comprise randomized BC and CD
loops. In another embodiment, the present invention comprise
randomized BC and DE loops. In another embodiment, the present
invention comprise randomized BC and EF loops. In another
embodiment, the present invention comprise randomized BC and FG
loops. In another embodiment, the present invention comprise
randomized CD and DE loops. In another embodiment, the present
invention comprise randomized CD and EF loops. In another
embodiment, the present invention comprise randomized CD and FG
loops. In another embodiment, the present invention comprise
randomized DE and EF loops. In another embodiment, the present
invention comprise randomized DE and FG loops. In another
embodiment, the present invention comprise randomized EF and FG
loops.
[0226] In another embodiment, the present invention comprise three
randomized loops. For example, in one embodiment, the present
invention comprise randomized AB, BC and CD loops. In another
embodiment, the present invention comprise randomized AB, BC and DE
loops. In another embodiment, the present invention comprise
randomized AB, BC and EF loops. In another embodiment, the present
invention comprise randomized AB, BC and FG loops. In another
embodiment, the present invention comprise randomized AB, CD and DE
loops. In another embodiment, the present invention comprise
randomized AB, CD and EF loops. In another embodiment, the present
invention comprise randomized AB, CD and FG loops. In another
embodiment, the present invention comprise randomized AB, DE and EF
loops. In another embodiment, the present invention comprise
randomized AB, DE and FG loops. In another embodiment, the present
invention comprise randomized AB, EF and FG loops. In another
embodiment, the present invention comprise randomized BC, CD and DE
loops. In another embodiment, the present invention comprise
randomized BC, CD and EF loops. In another embodiment, the present
invention comprise randomized BC, CD and FG loops. In another
embodiment, the present invention comprise randomized BC, DE and EF
loops. In another embodiment, the present invention comprise
randomized BC, DE and FG loops. In another embodiment, the present
invention comprise randomized BC, EF and FG loops. In another
embodiment, the present invention comprise randomized CD, DE and EF
loops. In another embodiment, the present invention comprise
randomized CD, DE and FG loops. In another embodiment, the present
invention comprise randomized CD, EF and FG loops. In another
embodiment, the present invention comprise randomized DE, EF and FG
loops.
[0227] In another embodiment, the present invention comprise four
randomized loops. In another embodiment, the present invention
comprise randomized AB, BC, CD and DE loops. In another embodiment,
the present invention comprise randomized AB, BC, CD and EF loops.
In another embodiment, the present invention comprise randomized
AB, BC, CD and FG loops. In another embodiment, the present
invention comprise randomized AB, CD, DE and EF loops. In another
embodiment, the present invention comprise randomized AB, CD, DE
and FG loops. In another embodiment, the present invention comprise
randomized AB, CD, EF and FG loops. In another embodiment, the
present invention comprise randomized AB, DE, EF and FG loops. In
another embodiment, the present invention comprise randomized BC,
CD, DE and EF loops. In another embodiment, the present invention
comprise randomized BC, CD, DE and FG loops. In another embodiment,
the present invention comprise randomized BC, DE, EF and FG loops.
In another embodiment, the present invention comprise randomized
CD, DE, EF and FG loops.
[0228] In another embodiment, the present invention comprise five
randomized loops. In another embodiment, the present invention
comprise randomized AB, BC, CD, DE, and EF loops. In another
embodiment, the present invention comprise randomized AB, BC, CD,
DE, and FG loops. In another embodiment, the present invention
comprise randomized AB, CD, DE, EF and FG loops. In another
embodiment, the present invention comprise randomized AB, BC, DE,
EF and FG loops. In another embodiment, the present invention
comprise randomized AB, BC, CD, EF, and FG loops. In another
embodiment, the present invention comprise randomized BC, CD, DE,
EF and FG loops.
[0229] In another embodiment, the present invention comprise 6
randomized loops. In one embodiment, the present invention comprise
randomized AB, BC, CD, DE, EF, and FG loops.
[0230] In a specific embodiment, the present invention comprise 3
randomized loops wherein the BC, DE, and FG loops are all
randomized. In another embodiment, protein the present invention
comprise at least one randomized loop wherein, at least the AB, at
least the CD, at least the EF loops are randomized. In a specific
embodiment, the present invention comprise 3 randomized loops
wherein the AB, CD, and EF loops are all randomized. In another
specific embodiment, the present invention comprise randomized
loops, wherein the AB, BC, CD, DE, EF, and FG loops are all
randomized.
[0231] In another embodiment, the present invention comprise
randomized loops wherein at least one, or at least two, or at least
three, or at least four, or at least five, or at least six loops
are not randomized.
[0232] In one embodiment, the present invention comprise at least
one randomized loop, wherein one loop is not randomized. In one
embodiment, the present invention comprise at least one randomized
loop, wherein the AB loop is not randomized. In another embodiment,
the present invention comprise at least one randomized loop,
wherein the BC loop is not randomized. In another embodiment, the
present invention comprise at least one randomized loop, wherein
the CD loop is not randomized. In another embodiment, the present
invention comprise at least one randomized loop, wherein the DE
loop is not randomized. In another embodiment, the present
invention comprise at least one randomized loop, wherein the EF
loop is not randomized. In another embodiment, the present
invention comprise at least one randomized loop, wherein the FG
loop is not randomized.
[0233] In another embodiment, the present invention comprise at
least one randomized loop wherein two loops are not randomized. In
one embodiment, the present invention comprise at least one
randomized loop wherein at least the AB and BC loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the AB and CD loops
are not randomized. In another embodiment, the present invention
comprise at least one randomized loop wherein at least the AB and
DE loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the AB and EF loops are not randomized. In another embodiment, the
present invention comprise at least one randomized loop wherein at
least the AB and FG loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the BC and CD loops are not randomized. In
another embodiment, the present invention comprise at least one
randomized loop wherein at least the BC and DE loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the BC and EF loops
are not randomized. In another embodiment, the present invention
comprise at least one randomized loop wherein at least the BC and
FG loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the CD and DE loops are not randomized. In another embodiment, the
present invention comprise at least one randomized loop wherein at
least the CD and EF loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the CD and FG loops are not randomized. In
another embodiment, the present invention comprise at least one
randomized loop wherein at least the DE and EF loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the DE and FG loops
are not randomized. In another embodiment, the present invention
comprise at least one randomized loop wherein at least the EF and
FG loops are not randomized.
[0234] In another embodiment, the present invention comprise at
least one randomized loop wherein three loops are not randomized.
In another embodiment, the present invention comprise at least one
randomized loop wherein at least the AB, BC, and CD loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the AB, BC, and DE
loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the AB, BC, and EF loops are not randomized. In another embodiment,
the present invention comprise at least one randomized loop wherein
at least the AB, BC, and FG loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the AB, CD, and DE loops are not randomized.
In another embodiment, the present invention comprise at least one
randomized loop wherein at least the AB, CD, and EF loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the AB, CD, and FG
loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the AB, DE, and EF loops are not randomized. In another embodiment,
the present invention comprise at least one randomized loop wherein
at least the AB, EF, and FG loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the BC, CD, and DE loops are not randomized.
In another embodiment, the present invention comprise at least one
randomized loop wherein at least the BC, CD, and EF loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the BC, CD, and FG
loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the BC, DE, and EF loops are not randomized. In another embodiment,
the present invention comprise at least one randomized loop wherein
at least the BC, EF, and FG loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the CD, DE, and EF loops are not randomized.
In another embodiment, the present invention comprise at least one
randomized loop wherein at least the CD, DE, and FG loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the CD, EF, and FG
loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the DE, EF, and FG loops are not randomized.
[0235] In another embodiment, the present invention comprise at
least one randomized loop, wherein four loops are not randomized.
In another embodiment, the present invention comprise at least one
randomized loop wherein at least the AB, BC, CD, and DE loops are
not randomized. In another embodiment, the present invention
comprise at least one randomized loop wherein at least the AB, BC,
CD, and EF loops are not randomized. In another embodiment, the
present invention comprise at least one randomized loop wherein at
least the AB, BC, CD, and FG loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the AB, CD, DE, and EF loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the AB, CD, DE, and
FG loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the AB, CD, EF, and FG loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the AB, DE, EF, and FG loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the BC, CD, DE and EF
loops are not randomized. In another embodiment, the present
invention comprise at least one randomized loop wherein at least
the BC, CD, DE and FG loops are not randomized. In another
embodiment, the present invention comprise at least one randomized
loop wherein at least the BC, DE, EF, and FG loops are not
randomized. In another embodiment, the present invention comprise
at least one randomized loop wherein at least the CD, DE, EF and FG
loops are not randomized.
[0236] In another embodiment, the present invention comprise at
least one randomized loop, wherein five loops are not randomized.
In another embodiment, the present invention comprise at least one
randomized loop wherein at least the AB, BC, CD, DE and EF loops
are not randomized. In another embodiment, the present invention
comprise at least one randomized loop wherein at least the AB, BC,
CD, DE and FG loops are not randomized. In another embodiment, the
present invention comprise at least one randomized loop wherein at
least the AB, CD, DE, EF and FG loops are not randomized. In
another embodiment, the present invention comprise at least one
randomized loop wherein at least the AB, BC, DE, EF and FG loops
are not randomized. In another embodiment, the present invention
comprise at least one randomized loop wherein at least the AB, BC,
CD, EF and FG loops are not randomized. In another embodiment, the
present invention comprise at least one randomized loop wherein at
least the BC, CD, DE, EF and FG loops are not randomized.
[0237] In another embodiment, the present invention comprise at
least one randomized loop, wherein six loops are not randomized. In
another embodiment, the present invention comprise at least one
randomized loop wherein at least the AB, BC, CD, DE, EF and FG
loops are not randomized.
[0238] The invention also provides scaffolds wherein the beta
strand regions are randomized wherein said beta strand randomized
scaffold exhibits a stability and specific target affinity at least
as high as the same scaffold prior to beta strand randomization
measured under similar conditions. In one embodiment, the present
invention comprise at least one, at least two, at least three, at
least four, at least five, or at least size beta strands are
randomized. In another embodiment, the present invention comprise
at least the A strand, or at least the B strand, or at least the C
strand, or at least the D strand, or at least the E strand, or at
least the F strand is randomized.
[0239] In another embodiment, the present invention comprise two
beta strands that are randomized. In another embodiment, the
present invention comprise a randomized A strand and B strand. In
another embodiment, the present invention comprise a randomized A
strand and C strand. In another embodiment, the present invention
comprise a randomized A strand and D strand. In another embodiment,
the present invention comprise a randomized A strand and E strand.
In another embodiment, the present invention comprise a randomized
A strand and F strand. In another embodiment, the present invention
comprise a randomized A strand and G strand. In another embodiment,
the present invention comprise a randomized B strand and C strand.
In another embodiment, the present invention comprise a randomized
B strand and D strand. In another embodiment, the present invention
comprise a randomized B strand and E strand. In another embodiment,
the present invention comprise a randomized B strand and F strand.
In another embodiment, the present invention comprise a randomized
B strand and G strand. In another embodiment, the present invention
comprise a randomized C strand and D strand. In another embodiment,
the present invention comprise a randomized C strand and E strand.
In another embodiment, the present invention comprise a randomized
C strand and F strand. In another embodiment, the present invention
comprise a randomized C strand and G strand. In another embodiment,
the present invention comprise a randomized D strand and E strand.
In another embodiment, the present invention comprise a randomized
D strand and F strand. In another embodiment, the present invention
comprise a randomized D strand and G strand. In another embodiment,
the present invention comprise a randomized E strand and F strand.
In another embodiment, the present invention comprise a randomized
E strand and G strand. In another embodiment, the present invention
comprise a randomized F strand and G strand.
[0240] In another embodiment, the present invention comprise three
beta strands that are randomized. In one embodiment, the present
invention comprise a randomized A, B, and C strand. In another
embodiment, the present invention comprise a randomized A, B, and D
strand. In another embodiment, the present invention comprise a
randomized A, B, and E strand. In another embodiment, the present
invention comprise a randomized A, B, and F strand. In another
embodiment, the present invention comprise a randomized A, B, and G
strand. In another embodiment, the present invention comprise a
randomized A, C, and D strand. In another embodiment, the present
invention comprise a randomized A, C, and E strand. In another
embodiment, the present invention comprise a randomized A, C, and F
strand. In another embodiment, the present invention comprise a
randomized A, C, and G strand. In another embodiment, the present
invention comprise a randomized A, D, and E strand. In another
embodiment, the present invention comprise a randomized A, D, and F
strand. In another embodiment, the present invention comprise a
randomized A, D, and G strand. In another embodiment, the present
invention comprise a randomized B, C, and D strand. In another
embodiment, the present invention comprise a randomized B, C, and E
strand. In another embodiment, the present invention comprise a
randomized B, C, and F strand. In another embodiment, the present
invention comprise a randomized B, C, and G strand. In another
embodiment, the present invention comprise a randomized B, D, and E
strand. In another embodiment, the present invention comprise a
randomized B, D, and F strand. In another embodiment, the present
invention comprise a randomized B, D, and G strand. In another
embodiment, the present invention comprise a randomized C, D, and E
strand. In another embodiment, the present invention comprise a
randomized C, D, and F strand. In another embodiment, the present
invention comprise a randomized C, D, and G strand. In another
embodiment, the present invention comprise a randomized C, E, and F
strand. In another embodiment, the present invention comprise a
randomized C, E, and G strand. In another embodiment, the present
invention comprise a randomized C, F, and G strand. In another
embodiment, the present invention comprise a randomized D, E, and F
strand. In another embodiment, the present invention comprise a
randomized D, F, and G strand. In another embodiment, the present
invention comprise a randomized E, F, and G strand.
[0241] In one embodiment the present invention comprise four beta
strands that are randomized. In another embodiment, the present
invention comprise a randomized A, B, C, and D strand. In another
embodiment, the present invention comprise a randomized A, B, C,
and E strand. In another embodiment, the present invention comprise
a randomized A, B, C, and F strand. In another embodiment, the
present invention comprise a randomized A, B, C, and G strand. In
another embodiment, the present invention comprise a randomized A,
C, D, and E strand. In another embodiment, the present invention
comprise a randomized A, C, D, and F strand. In another embodiment,
the present invention comprise a randomized A, C, D, and G strand.
In another embodiment, the present invention comprise a randomized
A, D, E, and F strand. In another embodiment, the present invention
comprise a randomized A, D, E, and G strand. In another embodiment,
the present invention comprise a randomized A, E, F, and G strand.
In another embodiment, the present invention comprise a randomized
B, C, D, and E strand. In another embodiment, the present invention
comprise a randomized B, C, D, and F strand. In another embodiment,
the present invention comprise a randomized B, C, D, and G strand.
In another embodiment, the present invention comprise a randomized
B, D, E, and F strand. In another embodiment, the present invention
comprise a randomized B, D, E, and G strand. In another embodiment,
the present invention comprise a randomized B, E, F, and G strand.
In another embodiment, the present invention comprise a randomized
C, D, E and F strand. In another embodiment, the present invention
comprise a randomized C, D, E, and G strand. In another embodiment,
the present invention comprise a randomized D, E, F, and G
strand.
[0242] In one embodiment, the present invention comprise five beta
strands that are randomized. In another embodiment, the present
invention comprise a randomized A, B, C, D, and E strand. In
another embodiment, the present invention comprise a randomized A,
B, C, D, and F strand. In another embodiment, the present invention
comprise a randomized A, B, C, D, and G strand. In another
embodiment, the present invention comprise a randomized A, C, D, E
and F strand. In another embodiment, the present invention comprise
a randomized A, C, D, E and G strand. In another embodiment, the
present invention comprise a randomized A, C, D, E and F strand. In
another embodiment, the present invention comprise a randomized A,
C, D, E, and G strand. In another embodiment, the present invention
comprise a randomized A, B, C, E and F strand. In another
embodiment, the present invention comprise a randomized A, B, C, E
and G strand. In another embodiment, the present invention comprise
a randomized A, B, C, D and F strand. In another embodiment, the
present invention comprise a randomized A, B, C, D and G strand. In
another embodiment, the present invention comprise a randomized B,
C, D, E, and F strand. In another embodiment, the present invention
comprise a randomized B, C, D, E, and G strand. In another
embodiment, the present invention comprise a randomized B, D, E, F
and G strand. In another embodiment, the present invention comprise
a randomized B, C, E, F and G strand. In another embodiment, the
present invention comprise a randomized B, C, D, F and G strand. In
another embodiment, the present invention comprise a randomized C,
D, E, F, and G strand.
[0243] In one embodiment, the present invention comprise six
randomized beta strands. In one embodiment, the present invention
comprise a randomized A, B, C, D, E, and F strand. In another
embodiment, the present invention comprise a randomized A, B, C, D,
E, and G strand. In another embodiment, the present invention
comprise a randomized A, C, D, E, F and G strand. In another
embodiment, the present invention comprise a randomized A, B, D, E,
F and G strand. In another embodiment, the present invention
comprise a randomized A, B, C, E, F and G strand. In another
embodiment, the present invention comprise a randomized A, B, C, D,
F and G strand. In another embodiment, the present invention
comprise a randomized A, B, C, D, E and G strand. In another
embodiment, the present invention comprise a randomized B, C, D, E,
F and G strand.
[0244] In one embodiment, the present invention comprise six
randomized beta strands. In one embodiment, the present invention
comprise a randomized A, B, C, D, E, F, and G strand.
[0245] The invention also provides protein scaffolds with loop
length diversity. In one embodiment, the present invention comprise
at least one loop comprising at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14,
at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, or at
least 30 amino acid residues. In another embodiment, protein the
present invention may comprise at least one loop comprising 1, 2,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In
another embodiment, the present invention may comprise at least
two, at least three, at least four, at least five or at least six
loops of the same length. In another embodiment, the present
invention may comprise at least two loops, at least three, at least
four, at least five or at least six loops of the different
lengths.
[0246] In another embodiment, the present invention vary from a
naturally occurring protein sequence by a deletion, substitution or
addition of at least one amino acid from the corresponding loop
sequences in the naturally occurring protein sequence. In one
embodiment, the present invention comprise a deletion, substitution
or addition of at least one amino acid in at least one, or at least
two, or at least three, or at least four, or at least five, or at
least six loop sequences from the corresponding naturally occurring
protein sequence. In one embodiment, the present invention comprise
a deletion, substitution or addition of at least 1, or at least 2,
or at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
11, or at least 12, or at least 13, or at least 14, or at least 15,
or at least 16, or at least 17, or at least 18, or at least 19, or
at least 20, or at least 21, or at least 22, or at least 23, or at
least 24, or at least 25, or at least 26, or at least 27, or at
least 28, or at least 29, or at least 30 amino acids in at least
one, or at least two, or at least three, or at least four, or at
least five, or at least six loop sequences from the corresponding
naturally occurring protein sequence. In another embodiment, the
present invention comprise a deletion, substitution or addition of
at least 1, at least 2, at least 3, at least 4 to about at least 8,
at least, at least 9, or at least 10, or at least 11, or at least
12, or at least 13, or at least 14, or at least 15, or at least 16,
or at least 17, or at least 18, or at least 19, or at least 20, or
at least 21, or at least 22, or at least 23, or at least 24, or at
least 25, or at least 26, or at least 27, or at least 28, or at
least 29, or at least 30 amino acids in at least one, or at least
two, or at least three, or at least four, or at least five, or at
least six loop sequences from the corresponding naturally occurring
protein sequence.
[0247] In one embodiment, the present invention comprise a
deletion, substitution or addition of at least one amino acid in
loop AB. In another embodiment the present invention comprise a
deletion, substitution or addition of at least 1, or at least 2, or
at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
11, or at least 12, or at least 13, or at least 14, or at least 15,
or at least 16, or at least 17, or at least 18, or at least 19, or
at least 20, or at least 21, or at least 22, or at least 23, or at
least 24, or at least 25, or at least 26, or at least 27, or at
least 28, or at least 29, or at least 30 amino acids in loop AB. In
another embodiment the present invention comprise a deletion,
substitution or addition of at least 1, at least 2, at least 3, at
least 4 to about at least 8, at least, at least 9, or at least 10,
or at least 11, or at least 12, or at least 13, or at least 14, or
at least 15, or at least 16, or at least 17, or at least 18, or at
least 19, or at least 20, or at least 21, or at least 22, or at
least 23, or at least 24, or at least 25, or at least 26, or at
least 27, or at least 28, or at least 29, or at least 30 amino
acids amino acids in loop AB.
[0248] In one embodiment, the present invention comprise a
deletion, substitution or addition of at least one amino acid in
loop BC. In another embodiment the present invention comprise a
deletion, substitution or addition of at least 1, or at least 2, or
at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
11, or at least 12, or at least 13, or at least 14, or at least 15,
or at least 16, or at least 17, or at least 18, or at least 19, or
at least 20, or at least 21, or at least 22, or at least 23, or at
least 24, or at least 25, or at least 26, or at least 27, or at
least 28, or at least 29, or at least 30 amino acids in loop BC. In
another embodiment the present invention comprise a deletion,
substitution or addition of at least 1, at least 2, at least 3, at
least 4 to about at least 8, at least, at least 9, or at least 10,
or at least 11, or at least 12, or at least 13, or at least 14, or
at least 15, or at least 16, or at least 17, or at least 18, or at
least 19, or at least 20, or at least 21, or at least 22, or at
least 23, or at least 24, or at least 25, or at least 26, or at
least 27, or at least 28, or at least 29, or at least 30 amino
acids amino acids in loop BC.
[0249] In one embodiment, the present invention comprise a
deletion, substitution or addition of at least one amino acid in
loop CD. In another embodiment the present invention comprise a
deletion, substitution or addition of at least 1, or at least 2, or
at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
11, or at least 12, or at least 13, or at least 14, or at least 15,
or at least 16, or at least 17, or at least 18, or at least 19, or
at least 20, or at least 21, or at least 22, or at least 23, or at
least 24, or at least 25, or at least 26, or at least 27, or at
least 28, or at least 29, or at least 30 amino acids in loop CD. In
another embodiment the present invention comprise a deletion,
substitution or addition of at least 1, at least 2, at least 3, at
least 4 to about at least 8, at least, at least 9, or at least 10,
or at least 11, or at least 12, or at least 13, or at least 14, or
at least 15, or at least 16, or at least 17, or at least 18, or at
least 19, or at least 20, or at least 21, or at least 22, or at
least 23, or at least 24, or at least 25, or at least 26, or at
least 27, or at least 28, or at least 29, or at least 30 amino
acids amino acids in loop CD.
[0250] In one embodiment, the present invention comprise a
deletion, substitution or addition of at least one amino acid in
loop DE. In another embodiment the present invention comprise a
deletion, substitution or addition of at least 1, or at least 2, or
at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
11, or at least 12, or at least 13, or at least 14, or at least 15,
or at least 16, or at least 17, or at least 18, or at least 19, or
at least 20, or at least 21, or at least 22, or at least 23, or at
least 24, or at least 25, or at least 26, or at least 27, or at
least 28, or at least 29, or at least 30 amino acids in loop DE. In
another embodiment the present invention comprise a deletion,
substitution or addition of at least 1, at least 2, at least 3, at
least 4 to about at least 8, at least, at least 9, or at least 10,
or at least 11, or at least 12, or at least 13, or at least 14, or
at least 15, or at least 16, or at least 17, or at least 18, or at
least 19, or at least 20, or at least 21, or at least 22, or at
least 23, or at least 24, or at least 25, or at least 26, or at
least 27, or at least 28, or at least 29, or at least 30 amino
acids amino acids in loop DE.
[0251] In one embodiment, the present invention comprise a
deletion, substitution or addition of at least one amino acid in
loop EF. In another embodiment the present invention comprise a
deletion, substitution or addition of at least 1, or at least 2, or
at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
11, or at least 12, or at least 13, or at least 14, or at least 15,
or at least 16, or at least 17, or at least 18, or at least 19, or
at least 20, or at least 21, or at least 22, or at least 23, or at
least 24, or at least 25, or at least 26, or at least 27, or at
least 28, or at least 29, or at least 30 amino acids in loop EF. In
another embodiment the present invention comprise a deletion,
substitution or addition of at least 1, at least 2, at least 3, at
least 4 to about at least 8, at least, at least 9, or at least 10,
or at least 11, or at least 12, or at least 13, or at least 14, or
at least 15, or at least 16, or at least 17, or at least 18, or at
least 19, or at least 20, or at least 21, or at least 22, or at
least 23, or at least 24, or at least 25, or at least 26, or at
least 27, or at least 28, or at least 29, or at least 30 amino
acids amino acids in loop EF.
[0252] In one embodiment, the present invention comprise a
deletion, substitution or addition of at least one amino acid in
loop FG. In another embodiment the present invention comprise a
deletion, substitution or addition of at least 1, or at least 2, or
at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
11, or at least 12, or at least 13, or at least 14, or at least 15,
or at least 16, or at least 17, or at least 18, or at least 19, or
at least 20, or at least 21, or at least 22, or at least 23, or at
least 24, or at least 25, or at least 26, or at least 27, or at
least 28, or at least 29, or at least 30 amino acids in loop FG. In
another embodiment the present invention comprise a deletion,
substitution or addition of at least 1, at least 2, at least 3, at
least 4 to about at least 8, at least, at least 9, or at least 10,
or at least 11, or at least 12, or at least 13, or at least 14, or
at least 15, or at least 16, or at least 17, or at least 18, or at
least 19, or at least 20, or at least 21, or at least 22, or at
least 23, or at least 24, or at least 25, or at least 26, or at
least 27, or at least 28, or at least 29, or at least 30 amino
acids amino acids in loop FG.
[0253] The invention also provides scaffolds comprising loop
sequence diversity. In one embodiment, the present invention
comprise at least 1, at least 2, at least 3, at least 4, at least 5
or at least 6 loops which comprise at least 1, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, at least 25, at least 26, at least 27, at least 28, at
least 29, or at least 30 positions that are randomized. In another
embodiment, the present invention comprise at least 1, at least 2,
at least 3, at least 4, at least 5 or at least 6 loops which
comprise at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7, at least 8, at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, at least 25, at
least 26, at least 27, at least 28, at least 29, or at least 30
positions that are randomized while further comprising at least 1,
at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 16, at least
17, at least 18, at least 19, at least 20, at least 21, at least
22, at least 23, at least 24, at least 25, at least 26, at least
27, at least 28, at least 29, or at least 30 positions that is held
constant. In another embodiment, the present invention comprise at
least 1, at least 2, at least 3, at least 4, at least 5 or at least
6 loops which comprise at least 1, at least 2, at least 3, at least
4, at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, or at
least 30 positions that is subjected to a restricted randomization.
In another embodiment, the present invention comprise at least 1,
at least 2, at least 3, at least 4, at least 5 or at least 6 loops
which comprise at least 1, at least 2, at least 3, at least 4, at
least 5 or at least 6 loops which comprise at least 1, at least 2,
at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, at least 25, at least 26, at least 27, at
least 28, at least 29, or at least 30 positions that is subjected
to a restricted randomization and further comprises at least 1, at
least 2, at least 3, at least 4, at least 5 or at least 6 loops
which comprise at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 11, at least 12, at least 13, at least 14, at least
15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, at least
25, at least 26, at least 27, at least 28, at least 29, or at least
30 positions that are held constant. In another embodiment, the
present invention comprise at least 1, at least 2, at least 3, at
least 4, at least 5 or at least 6 loops which comprise at least 1,
at least 2, at least 3, at least 4, at least 5 or at least 6 loops
which comprise at least 1, at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 11, at least 12, at least 13, at least 14, at least
15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, at least
25, at least 26, at least 27, at least 28, at least 29, or at least
30 positions that are subjected to a restricted randomization and
further comprises at least 1, at least 2, at least 3, at least 4,
at least 5 or at least 6 loops which comprise at least 1, at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, at least 25, at least 26, at least 27, at
least 28, at least 29, or at least 30 positions that are randomized
and at least 1, at least 2, at least 3, at least 4, at least 5 or
at least 6 loops which comprise at least 1, at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, at least 25, at least 26, at least 27, at least 28, at
least 29, or at least 30 positions that are held constant.
[0254] The invention also provides scaffolds comprising loop
sequence diversity. In one embodiment, the present invention
comprise at least one loop which contains at least one position
that is randomized. In another embodiment, the present invention
comprise at least one loop which comprises at least one position
that is randomized while further comprising at least one position
that is held constant. In another embodiment, the present invention
comprise a loop which comprises at least one position that is
subjected to a restricted randomization. In another embodiment, the
present invention comprise at least one loop which comprises at
least one position that is subjected to a restricted randomization
and further comprises at least one position that is held constant.
In another embodiment, the present invention comprise at least one
loop which comprises at least one position that is subjected to a
restricted randomization and further comprises at least one
position that is randomized and at least one position that is held
constant.
[0255] In one embodiment, the present invention comprise at least
one, at least two, at least three, at least four, at least five, or
at least six loops randomized for length and diversity. In one
embodiment, the present invention comprise an AB loop randomized
for sequence length and diversity. In another embodiment, the
present invention comprise a BC loop randomized for sequence length
and diversity. the present invention comprise a CD loop randomized
for sequence length and diversity. the present invention comprise a
DE loop randomized for sequence length and diversity. the present
invention comprise an EF loop randomized for sequence length and
diversity. the present invention comprise a FG loop randomized for
sequence length and diversity.
[0256] In another embodiment, the present invention comprise AB and
BC loops randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB and CD loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB and DE loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC and CD loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC and DE loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise CD and DE loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise CD and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise CD and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise DE and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise DE and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise EF and FG loops
randomized for sequence length and diversity.
[0257] In another embodiment, the present invention comprise AB, BC
and CD loops randomized for sequence length and diversity. In
another embodiment, the present invention comprise AB, BC and DE
loops randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, BC and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, BC and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, CD and DE loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, CD and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, CD and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, DE and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, DE and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, EF and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, CD and DE loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, CD and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, CD and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, DE and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, DE and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, EF and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise CD, DE and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise CD, DE and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise CD, EF and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise DE, EF and FG loops
randomized for sequence length and diversity.
[0258] In another embodiment, the present invention comprise AB,
BC, CD, and DE loops randomized for sequence length and diversity.
In another embodiment, the present invention comprise AB, BC, CD,
and EF loops randomized for sequence length and diversity. In
another embodiment, the present invention comprise AB, BC, CD, and
FG loops randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, CD, DE, and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, CD, DE, and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, CD, EF, and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise AB, DE, EF, and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, CD, DE and EF loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, CD, DE and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, CD, EF and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise BC, DE, EF and FG loops
randomized for sequence length and diversity. In another
embodiment, the present invention comprise CD, DE, EF, and FG loops
randomized for sequence length and diversity.
[0259] In another embodiment, the present invention comprise AB,
BC, CD, DE, and EF loops randomized for sequence length and
diversity. In another embodiment, the present invention comprise
AB, BC, CD, DE, and FG loops randomized for sequence length and
diversity. In another embodiment, the present invention comprise
AB, CD, DE, EF and FG loops randomized for sequence length and
diversity. In another embodiment, the present invention comprise
AB, BC, DE, EF and FG loops randomized for sequence length and
diversity. In another embodiment, the present invention comprise
AB, BC, CD, EF and FG loops randomized for sequence length and
diversity. In another embodiment, the present invention comprise
AB, BC, CD, EF, and FG loops randomized for sequence length and
diversity. In another embodiment, the present invention comprise
AB, BC, CD, DE, and EF loops randomized for sequence length and
diversity. In another embodiment, the present invention comprise
BC, CD, DE, EF and FG loops randomized for sequence length and
diversity.
6.8 Scaffold Conjugates
[0260] The present invention encompasses the use of scaffolds
conjugated or fused to one or more moieties, including but not
limited to, peptides, polypeptides, proteins, fusion proteins,
nucleic acid molecules, small molecules, mimetic agents, synthetic
drugs, inorganic molecules, and organic molecules. The present
invention encompasses the use of scaffolds recombinantly fused or
chemically conjugated (including both covalent and non-covalent
conjugations) to a heterologous protein or polypeptide (or fragment
thereof, to a polypeptide of at least 10, at least 20, at least 30,
at least 40, at least 50, at least 60, at least 70, at least 80, at
least 90 or at least 100 amino acids) to generate fusion proteins.
The fusion does not necessarily need to be direct, but may occur
through linker sequences described herein. For example, scaffolds
may be used to target heterologous polypeptides to particular cell
types, either in vitro or in vivo, by fusing or conjugating the
scaffolds to antibodies specific for particular cell surface
receptors. Scaffolds fused or conjugated to heterologous
polypeptides may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
International publication No. WO 93/21232; European Patent No. EP
439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat.
No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et
al., 1991, J. Immunol. 146:2446-2452, which are incorporated by
reference in their entireties.
[0261] Additional fusion proteins comprising scaffolds of the
invention may be generated through the techniques of
gene-shuffling, motif-shuffling, exon-shuffling, and/or
codon-shuffling (collectively referred to as "DNA shuffling"). DNA
shuffling may be employed to alter the activities of scaffolds of
the invention (e.g., scaffolds with higher affinities and lower
dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793;
5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al.,
1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends
Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol.
287:265-76; and Lorenzo and Blasco, 1998, Biotechniques
24(2):308-313 (each of these patents and publications are hereby
incorporated by reference in its entirety). Scaffolds, or the
encoded scaffolds thereof, may be altered by being subjected to
random mutagenesis by error-prone PCR, random nucleotide insertion
or other methods prior to recombination. One or more portions of a
polynucleotide encoding a scaffold, which bind to a specific target
may be recombined with one or more components, motifs, sections,
parts, domains, fragments, etc. of one or more heterologous
molecules.
[0262] Moreover, the scaffolds of the invention can be fused to
marker sequences, such as a peptide to facilitate purification. In
some embodiments, the marker amino acid sequence is a
hexa-histidine peptide (his-tag), such as the tag provided in a pQE
vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311),
among others, many of which are commercially available. As
described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA
86:821-824, for instance, hexa-histidine provides for convenient
purification of the fusion protein. Other peptide tags useful for
purification include, but are not limited to, the hemagglutinin
"HA" tag, which corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767)
and the "flag" tag.
[0263] In other embodiments, scaffolds of the invention, analogs or
derivatives thereof may be conjugated to a diagnostic or detectable
agent. Such scaffolds can be useful for monitoring or prognosing
the development or progression of a disease as part of a clinical
testing procedure, such as determining the efficacy of a particular
therapy. Such diagnosis and detection can be accomplished by
coupling the scaffold to detectable substances including, but not
limited to various enzymes, such as but not limited to horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; prosthetic groups, such as but not limited to
streptavidin/biotin and avidin/biotin; fluorescent materials, such
as but not limited to, umbelliferone, fluorescein, fluorescein
isothiocynate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride or phycoerythrin; luminescent materials, such as,
but not limited to, luminol; bioluminescent materials, such as but
not limited to, luciferase, luciferin, and aequorin; radioactive
materials, such as but not limited to iodine (.sup.131I, .sup.125I,
.sup.123I, .sup.121I), carbon (.sup.14C) sulfur (.sup.35S), tritium
(3H), indium (.sup.115In, .sup.113In, .sup.112In, .sup.111In), and
technetium (.sup.99Tc), thallium (.sup.201Ti), gallium (.sup.68Ga,
.sup.67Ga), palladium (.sup.103Pd), molybdenum (.sup.99Mo), xenon
(.sup.133Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu,
.sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb, .sup.166Ho,
.sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re, .sup.142Pr,
.sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr,
.sup.32P, .sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se,
.sup.113Sn, and .sup.117Tn; positron emitting metals using various
positron emission tomographies, nonradioactive paramagnetic metal
ions, and molecules that are radiolabelled or conjugated to
specific radioisotopes.
[0264] The present invention further encompasses uses of scaffolds
conjugated to a therapeutic moiety. A scaffold may be conjugated to
a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Therapeutic moieties include,
but are not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and
doxorubicin), antibiotics (e.g., dactinomycin (formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),
Auristatin molecules (e.g., auristatin PHE, bryostatin 1, and
solastatin 10; see Woyke et al., Antimicrob. Agents Chemother.
46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother.
45:3580-4 (2001), Mohammad et al., Anticancer Drugs 12:735-40
(2001), Wall et al., Biochem. Biophys. Res. Commun. 266:76-80
(1999), Mohammad et al., Int. J. Oncol. 15:367-72 (1999), all of
which are incorporated herein by reference), hormones (e.g.,
glucocorticoids, progestins, androgens, and estrogens), DNA-repair
enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors
(e.g., compound ST1571, imatinib mesylate (Kantarjian et al., Clin
Cancer Res. 8(7):2167-76 (2002)), cytotoxic agents (e.g.,
paclitaxel, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, procaine, tetracaine, lidocaine,
propranolol, and puromycin and analogs or homologs thereof) and
those compounds disclosed in U.S. Pat. Nos. 6,245,759, 6,399,633,
6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372,
6,057,300 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844,
5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239,
5,587,459), farnesyl transferase inhibitors (e.g., R115777,
BMS-214662 and those disclosed by, for example, U.S. Pat. Nos.
6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387,
6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905,
6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501,
6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865,
6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096,
6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295,
6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935,
6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and
6,040,305), topoisomerase inhibitors (e.g., camptothecin;
irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI
147211); DX-8951f; IST-622; rubitecan; pyrazoloacridine; XR-5000;
saintopin; UCE6; UCE1022; TAN-1518A; TAN-1518B; KT6006; KT6528;
ED-110; NB-506; ED-110; NB-506; and rebeccamycin); bulgarein; DNA
minor groove binders such as Hoescht dye 33342 and Hoechst dye
33258; nitidine; fagaronine; epiberberine; coralyne;
beta-lapachone; BC-4-1; bisphosphonates (e.g., alendronate,
cimadronte, clodronate, tiludronate, etidronate, ibandronate,
neridronate, olpandronate, risedronate, piridronate, pamidronate,
zolendronate) HMG-CoA reductase inhibitors, (e.g., lovastatin,
simvastatin, atorvastatin, pravastatin, fluvastatin, statin,
cerivastatin, lescol, lupitor, rosuvastatin and atorvastatin) and
pharmaceutically acceptable salts, solvates, clathrates, and
prodrugs thereof. See, e.g., Rothenberg, M. L., Annals of Oncology
8:837-855 (1997); and Moreau, P., et al., J. Med. Chem.
41:1631-1640 (1998)), antisense oligonucleotides (e.g., those
disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,
5,734,033, and 5,618,709), immunomodulators (e.g., antibodies and
cytokines), antibodies, and adenosine deaminase inhibitors (e.g.,
Fludarabine phosphate and 2-Chlorodeoxyadenosine). Examples include
paclitaxel, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C and cisdichlorodiamine
platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, mithramycin, and
anthramycin (AMC)), Auristatin molecules (e.g., auristatin PHE,
bryostatin 1, solastatin 10, see Woyke et al., Antimicrob. Agents
Chemother. 46:3802-8 (2002), Woyke et al., Antimicrob. Agents
Chemother. 45:3580-4 (2001), Mohammad et al., Anticancer Drugs
12:735-40 (2001), Wall et al., Biochem. Biophys. Res. Commun.
266:76-80 (1999), Mohammad et al., Int. J. Oncol. 15:367-72 (1999),
all of which are incorporated herein by reference), anti-mitotic
agents (e.g., vincristine and vinblastine), hormones (e.g.,
glucocorticoids, progestatins, androgens, and estrogens),
DNA-repair enzyme inhibitors (e.g., etoposide or topotecan), kinase
inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et
al., Clin Cancer Res. 8(7):2167-76 (2002)), and those compounds
disclosed in U.S. Pat. Nos. 6,245,759, 6,399,633, 6,383,790,
6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300,
6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995,
5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, 5,587,459),
farnesyl transferase inhibitors (e.g., R115777, BMS-214662, and
those disclosed by, for example, U.S. Pat. Nos. 6,458,935,
6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145,
6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747,
6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363,
6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865, 6,228,856,
6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096, 6,159,984,
6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295, 6,103,723,
6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935, 6,066,738,
6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305),
topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38;
topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f;
IST-622; rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6;
UCE1022; TAN-1518A; TAN-1518B; KT6006; KT6528; ED-110; NB-506;
ED-110; NB-506; and rebeccamycin; bulgarein; DNA minor groove
binders such as Hoescht dye 33342 and Hoechst dye 33258; nitidine;
fagaronine; epiberberine; coralyne; beta-lapachone; BC-4-1; and
pharmaceutically acceptable salts, solvates, clathrates, and
prodrugs thereof. See, e.g., Rothenberg, M. L., Annals of Oncology
8:837-855 (1997); and Moreau, P., et al., J. Med. Chem.
41:1631-1640 (1998)), antisense oligonucleotides (e.g., those
disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,
5,734,033, and 5,618,709), immunomodulators (e.g., antibodies and
cytokines), antibodies (e.g., rituximab (Rituxan.RTM.),
calicheamycin (Mylotarg.RTM., ibritumomab tiuxetan (Zevalin.RTM.),
and tositumomab (Bexxar.RTM.)), and adenosine deaminase inhibitors
(e.g., Fludarabine phosphate and 2-Chlorodeoxyadenosine).
[0265] Further, a scaffold may be conjugated to a therapeutic
moiety or drug moiety that modifies a given biological response.
Therapeutic moieties or drug moieties are not to be construed as
limited to classical chemical therapeutic agents. For example, the
drug moiety may be a protein or polypeptide possessing a desired
biological activity. Such proteins may include, for example, a
toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin,
or diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator, an
apoptotic agent, e.g., TNF-.alpha., TNF-.beta., AIM I (see,
International publication No. WO 97/33899), AIM II (see,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., 1994, J. Immunol., 6:1567-1574), and VEGI (see,
International publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin, endostatin or a
component of the coagulation pathway (e.g., tissue factor); or, a
biological response modifier such as, for example, a lymphokine
(e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), and granulocyte colony stimulating factor
("G-CSF")), a growth factor (e.g., growth hormone ("GH")), or a
coagulation agent (e.g., calcium, vitamin K, tissue factors, such
as but not limited to, Hageman factor (factor XII),
high-molecular-weight kininogen (HMWK), prekallikrein (PK),
coagulation proteins-factors II (prothrombin), factor V, XIIa,
VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid. fibrinopeptides A
and B from the .alpha. and .beta. chains of fibrinogen, fibrin
monomer).
[0266] Moreover, a scaffold can be conjugated to therapeutic
moieties such as a radioactive metal ion, such as alpha-emitters
such as .sup.213Bi or macrocyclic chelators useful for conjugating
radiometal ions, including but not limited to, .sup.131In,
.sup.131LU, .sup.131Y, .sup.131Ho, .sup.131Sm, to polypeptides. In
certain embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraa-cetic acid
(DOTA) which can be attached to the scaffold via a linker molecule.
Such linker molecules are commonly known in the art and described
in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson
et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al.,
1999, Nucl. Med. Biol. 26(8):943-50, each incorporated by reference
in their entireties.
[0267] Techniques for conjugating therapeutic moieties to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies 84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol.
Rev. 62:119-58. Similar approaches may be adapted for use with
scaffolds of the invention.
[0268] The therapeutic moiety or drug conjugated to a scaffold of
the invention should be chosen to achieve the desired prophylactic
or therapeutic effect(s) for a particular disorder in a subject. A
clinician or other medical personnel should consider the following
when deciding on which therapeutic moiety or drug to conjugate to a
scaffold: the nature of the disease, the severity of the disease,
and the condition of the subject.
[0269] Scaffolds of the invention may also be attached to solid
supports, which are particularly useful for immunoassays or
purification of the target antigen. Such solid supports include,
but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene.
6.9 Production
[0270] Recombinant expression of a scaffold of the invention
requires construction of an expression vector containing a
polynucleotide that encodes the scaffold. Once a polynucleotide
encoding a scaffold has been obtained, the vector for the
production of scaffold may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods
for preparing a protein by expressing a polynucleotide containing a
scaffold encoding nucleotide sequence are described herein. Methods
that are well known to those skilled in the art can be used to
construct expression vectors containing scaffold polypeptide coding
sequences and appropriate transcriptional and translational control
signals. These methods include, for example, in vitro recombinant
DNA techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding a scaffold of the
invention, operably linked to a promoter.
[0271] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce a scaffold of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding a scaffold of the invention, operably linked to a
heterologous promoter. Suitable host cells include, but are not
limited to, microorganisms such as bacteria (e.g., E. coli and B.
subtilis).
[0272] A variety of host-expression vector systems may be utilized
to express the scaffolds of the invention. Such host-expression
systems represent vehicles by which the coding sequences of
interest may be produced and subsequently purified, but also
represent cells which may, when transformed or transfected with the
appropriate nucleotide coding sequences, express a scaffold of the
invention in situ. These include but are not limited to
microorganisms such as bacteria (e.g., E. coli and B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing scaffold coding sequences;
yeast (e.g., Saccharomyces, Pichia) transformed with recombinant
yeast expression vectors containing scaffold coding sequences;
insect cell systems infected with recombinant virus expression
vectors (e.g., baculovirus) containing scaffold coding sequences;
plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing scaffold coding sequences; or
mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0273] Expression vectors containing inserts of a gene encoding a
scaffold of the invention can be identified by three general
approaches: (a) nucleic acid hybridization, (b) presence or absence
of "marker" gene functions, and (c) expression of inserted
sequences. In the first approach, the presence of a gene encoding a
peptide, polypeptide, protein or a fusion protein in an expression
vector can be detected by nucleic acid hybridization using probes
comprising sequences that are homologous to an inserted gene
encoding the peptide, polypeptide, protein or the fusion protein,
respectively. In the second approach, the recombinant vector/host
system can be identified and selected based upon the presence or
absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, transformation phenotype,
occlusion body formation in baculovirus, etc.) caused by the
insertion of a nucleotide sequence encoding an antibody or fusion
protein in the vector. For example, if the nucleotide sequence
encoding the scaffold is inserted within the marker gene sequence
of the vector, recombinants containing the gene encoding the
scaffold insert can be identified by the absence of the marker gene
function. In the third approach, recombinant expression vectors can
be identified by assaying the gene product (e.g., scaffold or
multimer thereof) expressed by the recombinant. Such assays can be
based, for example, on the physical or functional properties of the
protein in in vitro assay systems, e.g., binding, agonistic or
antagonistic properties of the scaffold.
[0274] In some embodiments the scaffolds of the invention may be
chemically synthesized at least partially. In other embodiments,
the scaffolds of the invention may be produced
semi-synthetically.
6.10 Scaffold Purification
[0275] Once a scaffold of the invention has been produced by
recombinant expression, it may be purified by any method known in
the art for purification of a protein, for example, by
chromatography (e.g., metal-chelate chromatography, ion exchange,
affinity, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins.
[0276] The highly stable nature of the scaffolds of the invention
allow for variations on purification schemes. For example, the
thermal stability exhibited by the scaffolds of the invention allow
for the heating of the crude lysate comprising the scaffolds to
remove the bulk of the host cell proteins by denaturation. In
another embodiment, the high protease resistance exhibited by the
scaffolds of the invention allow for the rapid degradation of host
cell proteins in crude lysates prior to any purification steps.
Also, the pH tolerance exhibited by the scaffolds of the invention
allow for the selective precipitation of host cell proteins in the
crude lysate by lowering or raising the pH prior to any
purification steps. In some embodiments, the purification of the
scaffolds of the invention are facilitated by a high temperature
shift, a protease treatment, a pH shift up or down, or a
combination of any of the above in an effort to remove bulk host
cell proteins from the crude lysate. In some embodiments, the
protein remaining after the heat denaturation, protease treatment,
of pH shift is at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% specific scaffold protein.
[0277] In some embodiments, methods of purifying the scaffolds
comprise lowering the pH of the crude lysate containing said
scaffold to about 6.5, or to about 6.0, or to about 5.5, or to
about 5.0, or to about 4.5 or to about 4.0, or to about 3.5, or to
about 3.0 or to about 2.5, or to about 2.0 in an effort to
precipitate the host cell protein. In other embodiments, methods of
purification comprise raising the pH of the crude lysate containing
said scaffold to about 8.0, or to about 8.5, or to about 9.0, or to
about 9.5, or to about 10.0, or to about 10.5, or to about 11.0, or
to about 11.5, or to about 12.0, or to about 12.5 in an effort to
precipitate the host cell protein.
6.11 Scalable Production of Scaffolds
[0278] In an effort to obtain large quantities, scaffolds of the
invention may be produced by a scalable process (hereinafter
referred to as "scalable process of the invention"). In some
embodiments, scaffolds may be produced by a scalable process of the
invention in the research laboratory that may be scaled up to
produce the scaffolds of the invention in analytical scale
bioreactors (for example, but not limited to 5 L, 10 L, 15 L, 30 L,
or 50 L bioreactors). In other embodiments, the scaffolds may be
produced by a scalable process of the invention in the research
laboratory that may be scaled up to produce the scaffolds of the
invention in production scale bioreactors (for example, but not
limited to 75 L, 100 L, 150 L, 300 L, or 500 L). In some
embodiments, the scalable process of the invention results in
little or no reduction in production efficiency as compared to the
production process performed in the research laboratory.
[0279] In some embodiments, the scalable process of the invention
produces scaffolds at production efficiency of about 1 g/L, about 2
g/L, about 3 g/L, about 5 g/L, about 7.5 g/L, about 10 g/L, about
12.5 g/L, about 15.0 g/L, about 17.5 g/L, about 20 g/L, about 25
g/L, about 30 g/L, or higher.
[0280] In other embodiments, the scalable process of the invention
produces scaffolds at a production efficiency of at least about 1
g/L, at least about 2 g/L, at least about 3 g/L, at least about 5
g/L, at least about 7.5 g/L, at least about 10 g/L, at least about
12.5 g/L, at least about 15 g/L, at least about 17.5 g/L, at least
about 20 g/L, at least about 25 g/L, at least about 30 g/L, or
higher.
[0281] In other embodiments, the scalable process of the invention
produces scaffolds at a production efficiency from about 10 g/L to
about 300 g/L, from about 10 g/L to about 250 g/L, from about 10
g/L to about 200 g/L, from about 10 g/L to about 175 g/L, from
about 10 g/L to about 150 g/L, from about 10 g/L to about 100 g/L,
from about 20 g/L to about 300 g/L, from about 20 g/L to about 250
g/L, from about 20 g/L to about 200 g/L, from 20 g/L to about 175
g/L, from about 20 g/L to about 150 g/L, from about 20 g/L to about
125 g/L, from about 20 g/L to about 100 g/L, from about 30 g/L to
about 300 g/L, from about 30 g/L to about 250 g/L, from about 30
g/L to about 200 g/L, from about 30 g/L to about 175 g/L, from
about 30 g/L to about 150 g/L, from about 30 g/L to about 125 g/L,
from about 30 g/L to about 100 g/L, from about 50 g/L to about 300
g/L, from about 50 g/L to about 250 g/L, from about 50 g/L to about
200 g/L, from 50 g/L to about 175 g/L, from about 50 g/L to about
150 g/L, from about 50 g/L to about 125 g/L, or from about 50 g/L
to about 100 g/L.
[0282] In some embodiments, the scalable process of the invention
produces multimeric scaffolds at production efficiency of about 10
mg/L, about 20 m/L, about 30 mg/L, about 50 mg/L, about 75 mg/L,
about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L,
about 200 mg/L, about 250 mg/L, about 300 mg/L or higher.
[0283] In other embodiments, the scalable process of the invention
produces multimeric scaffolds at a production efficiency of at
least about 10 mg/L, at least about 20 m/L, at least about 30 mg/L,
at least about 50 mg/L, at least about 75 mg/L, at least about 100
mg/L, at least about 125 mg/L, at least about 150 mg/L, at least
about 175 mg/L, at least about 200 mg/L, at least about 250 mg/L,
at least about 300 mg/L or higher.
[0284] In other embodiments, the scalable process of the invention
produces multimeric scaffolds at a production efficiency from about
10 mg/L to about 300 mg/L, from about 10 mg/L to about 250 mg/L,
from about 10 mg/L to about 200 mg/L, from about 10 mg/L to about
175 mg/L, from about 10 mg/L to about 150 mg/L, from about 10 mg/L
to about 100 mg/L, from about 20 mg/L to about 300 mg/L, from about
20 mg/L to about 250 mg/L, from about 20 mg/L to about 200 mg/L,
from 20 mg/L to about 175 mg/L, from about 20 mg/L to about 150
mg/L, from about 20 mg/L to about 125 mg/L, from about 20 mg/L to
about 100 mg/L, from about 30 mg/L to about 300 mg/L, from about 30
mg/L to about 250 mg/L, from about 30 mg/L to about 200 mg/L, from
about 30 mg/L to about 175 mg/L, from about 30 mg/L to about 150
mg/L, from about 30 mg/L to about 125 mg/L, from about 30 mg/L to
about 100 mg/L, from about 50 mg/L to about 300 mg/L, from about 50
mg/L to about 250 mg/L, from about 50 mg/L to about 200 mg/L, from
50 mg/L to about 175 mg/L, from about 50 mg/L to about 150 mg/L,
from about 50 mg/L to about 125 mg/L, or from about 50 mg/L to
about 100 mg/L.
6.12 Production of Secreted Scaffolds
[0285] The invention also provides methods for the production of
scaffolds intracellularly or as a secreted form. In some
embodiments, the secreted scaffold is produced at levels described
herein. In other embodiments, secreted scaffolds are properly
folded and fully functional. In other embodiments, the production
of secreted scaffolds comprises the use of a Ptac promoter. In
other embodiments, the production of secreted scaffolds comprises
the use of a oppA signal. In yet other embodiments, the secreted
scaffold is expressed in a prokaryotic host cell. In further
embodiments, the scaffold is secreted into the periplasmic space of
a prokaryotic host cell. In yet other embodiments, the scaffold is
secreted directly into the media. In yet further embodiments,
scaffolds may be screened from crude cell culture media or
periplasm extracts.
[0286] The invention also provides methods for the secretion of
tandem proteins or fusions using protein scaffolds. In some
embodiments, scaffolds of the invention may act as carrier
molecules for the secretion of peptides and/or proteins into the
cell culture media or periplasmic space of a prokaryotic cell.
[0287] In another embodiment, methods of purifying scaffolds of the
invention comprise heating the crude lysate comprising said
scaffold to 70.degree. C. for 15 min and subsequently removing
aggregated compounds by centrifugation. In other embodiments,
methods of purifying scaffolds of the invention comprise heating
the crude lysate comprising said scaffold to about 50.degree. C.,
about 55.degree. C., about 60.degree. C., about 65.degree. C.,
about 70.degree. C., about 75.degree. C., about 80.degree. C.,
about 85.degree. C., or about 90.degree. C. and subsequently
removing aggregated compounds by centrifugation. In other
embodiments, methods of purifying scaffolds of the invention
comprise heating the crude lysate for at least about 1 min, about 2
min, about 3 min, about 4 min, about 5 min, about 6 min, about 7
min, about 8 min, about 9 min, about 10 min, about 11 min, about 12
min, about 13 min, about 14 min, about 15 min, about 20 min, or
about 30 min and subsequently removing aggregated compounds by
centrifugation.
[0288] In another specific embodiment, methods of purifying
scaffolds of the invention comprise shifting the pH of the crude
lysate to 3.0 and heating the crude lysate to comprising said
scaffold 70.degree. C. for 15 min and subsequently removing
aggregated compounds by centrifugation.
6.13 Assaying Scaffolds
[0289] The scaffolds of the invention may be assayed for specific
binding to a target by any method known in the art. Representative
assays which can be used, include but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitation reactions, gel diffusion precipitin
reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, to name but a few. Such assays are routine and known
in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York).
[0290] ELISAs comprise preparing antigen (e.g. a scaffold), coating
the well of a 96 well microtiter plate with the antigen, adding the
epitope binding protein of interest (e.g. a scaffold specific
antibody) conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the epitope binding protein of
interest does not have to be conjugated to a detectable compound;
instead, a second antibody (which recognizes the protein of
interest) conjugated to a detectable compound may be added to the
well. Further, instead of coating the well with the antigen, the
protein of interest may be coated to the well. In this case, a
second antibody conjugated to a detectable compound may be added
following the addition of the antigen of interest to the coated
well.
[0291] One of skill in the art would be knowledgeable as to the
parameters that can be modified to increase the signal detected as
well as other variations of ELISAs known in the art. For further
discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York at 11.2.1.
[0292] The binding affinity and other binding properties of a
scaffold to an antigen may be determined by a variety of in vitro
assay methods known in the art including for example, equilibrium
methods (e.g., enzyme-linked immunoabsorbent assay (ELISA; or
radioimmunoassay (RIA)), or kinetics (e.g., BIACORE.RTM. analysis),
and other methods such as indirect binding assays, competitive
binding assays fluorescence resonance energy transfer (FRET), gel
electrophoresis and chromatography (e.g., gel filtration). These
and other methods may utilize a label on one or more of the
components being examined and/or employ a variety of detection
methods including but not limited to chromogenic, fluorescent,
luminescent, or isotopic labels. A detailed description of binding
affinities and kinetics can be found in Paul, W. E., ed.,
Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia
(1999).
[0293] The stability of scaffolds of the invention may be increased
by many different approaches. In one embodiment, the scaffolds of
the invention comprise a non-naturally occurring disulfide bond, as
described herein. In another embodiment, the scaffolds of the
invention comprise an elongation of the N and/or C terminal
regions. In another embodiment, the scaffolds of the invention
comprise an addition, deletion or substitution of at least one
amino acid residue to adjust the surface charge of the scaffold. In
another embodiment, the scaffolds of the invention comprise an
alteration to increase serum half-life, as described herein. In yet
another embodiment, the scaffolds of the invention comprise an
addition, deletion or substitution of at least one amino acid
residue to stabilize the hydrophobic core of the scaffold.
[0294] The stability of scaffolds of the invention may be assessed
by many different techniques. A selection of techniques know in the
art include melting temperature, Differential scanning calorimetry
(DSC), Circular Dichroism (CD), Polyacrylamide gel electrophoresis
(PAGE), protease resistance, Isothermal calorimetry (ITC), nuclear
magnetic resonance (NMR), internal fluorescence, and biological
activity. In one embodiment, engineered scaffolds of the invention
exhibit increased stability compared to the same scaffold prior to
engineering.
6.14 Pharmaceutical Compositions
[0295] In another aspect, the present invention provides a
composition, for example, but not limited to, a pharmaceutical
composition, containing one or a combination of scaffolds or target
binding proteins of the present invention, formulated together with
a pharmaceutically acceptable carrier. Such compositions may
include one or a combination of, for example, but not limited to
two or more different scaffolds of the invention. For example, a
pharmaceutical composition of the invention may comprise a
combination of scaffolds that bind to different epitopes on the
target antigen or that have complementary activities.
[0296] Pharmaceutical compositions of the invention also can be
administered in combination therapy, such as, combined with other
agents. For example, the combination therapy can include a scaffold
of the present invention combined with at least one other therapy
wherein the therapy may be immunotherapy, chemotherapy, radiation
treatment, or drug therapy.
[0297] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. Examples of such
salts include acid addition salts and base addition salts. Acid
addition salts include those derived from nontoxic inorganic acids,
such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium and the like, as well
as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0298] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0299] Examples of suitable aqueous and non-aqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0300] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0301] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
suitable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0302] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0303] In one embodiment the compositions (e.g., liquid
formulations) of the invention are pyrogen-free formulations which
are substantially free of endotoxins and/or related pyrogenic
substances. Endotoxins include toxins that are confined inside a
microorganism and are released when the microorganisms are broken
down or die. Pyrogenic substances also include fever-inducing,
thermostable substances (glycoproteins) from the outer membrane of
bacteria and other microorganisms. Both of these substances can
cause fever, hypotension and shock if administered to humans. Due
to the potential harmful effects, it is advantageous to remove even
low amounts of endotoxins from intravenously administered
pharmaceutical drug solutions. The Food & Drug Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose
per kilogram body weight in a single one hour period for
intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight it is advantageous
to remove even trace amounts of endotoxin. In one embodiment,
endotoxin and pyrogen levels in the composition are less then 10
EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1
EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg. In
another embodiment, endotoxin and pyrogen levels in the composition
are less then about 10 EU/mg, or less then about 5 EU/mg, or less
then about 1 EU/mg, or less then about 0.1 EU/mg, or less then
about 0.01 EU/mg, or less then about 0.001 EU/mg.
6.15 Dosing/Administration
[0304] To prepare pharmaceutical or sterile compositions including
a scaffold of the invention, scaffold is mixed with a
pharmaceutically acceptable carrier or excipient. Formulations of
therapeutic and diagnostic agents can be prepared by mixing with
physiologically acceptable carriers, excipients, or stabilizers in
the form of, e.g., lyophilized powders, slurries, aqueous
solutions, lotions, or suspensions (see, e.g., Hardman, et al.
(2001) Goodman and Gilman's The Pharmacological Basis of
Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000)
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker,
NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY;
Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel
Dekker, Inc., New York, N.Y.).
[0305] Selecting an administration regimen for a therapeutic
depends on several factors, including the serum or tissue turnover
rate of the entity, the level of symptoms, the immunogenicity of
the entity, and the accessibility of the target cells in the
biological matrix. In certain embodiments, an administration
regimen maximizes the amount of therapeutic delivered to the
patient consistent with an acceptable level of side effects.
Accordingly, the amount of biologic delivered depends in part on
the particular entity and the severity of the condition being
treated. Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)
(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New
Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh,
et al. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000)
New Engl. J. Med. 343:1594-1602).
[0306] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0307] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0308] Scaffolds of the invention can be provided by continuous
infusion, or by doses at intervals of, e.g., one day, one week, or
1-7 times per week. Doses may be provided intravenously,
subcutaneously, topically, orally, nasally, rectally,
intramuscular, intracerebrally, or by inhalation. A specific dose
protocol is one involving the maximal dose or dose frequency that
avoids significant undesirable side effects. A total weekly dose
may be at least 0.05 .mu.g/kg body weight, at least 0.2 .mu.g/kg,
at least 0.5 .mu.g/kg, at least 1 .mu.g/kg, at least 10 .mu.g/kg,
at least 100 .mu.g/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at
least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or at least
50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J. Med.
349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;
Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456;
Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144).
The desired dose of a small molecule therapeutic, e.g., a peptide
mimetic, protein scaffold, natural product, or organic chemical, is
about the same as for an antibody or polypeptide, on a moles/kg
body weight basis. The desired plasma concentration of a small
molecule or scaffold therapeutic is about the same as for an
antibody, on a moles/kg body weight basis. The dose may be at least
15 .mu.g, at least 20 .mu.g, at least 25 .mu.g, at least 30 .mu.g,
at least 35 .mu.g, at least 40 .mu.g, at least 45 .mu.g, at least
50 .mu.g, at least 55 .mu.g, at least 60 .mu.g, at least 65 .mu.g,
at least 70 .mu.g, at least 75 .mu.g, at least 80 .mu.g, at least
85 .mu.g, at least 90 .mu.g, at least 95 .mu.g, or at least 100
.mu.g. The doses administered to a subject may number at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.
[0309] For scaffolds of the invention, the dosage administered to a
patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body
weight. The dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001
mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg,
0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and
0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001
to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to
0.10 mg/kg of the patient's body weight.
[0310] The dosage of the scaffolds of the invention may be
calculated using the patient's weight in kilograms (kg) multiplied
by the dose to be administered in mg/kg. The dosage of the
scaffolds of the invention may be 150 .mu.g/kg or less, 125
.mu.g/kg or less, 100 .mu.g/kg or less, 95 .mu.g/kg or less, 90
.mu.g/kg or less, 85 .mu.g/kg or less, 80 .mu.g/kg or less, 75
.mu.g/kg or less, 70 .mu.g/kg or less, 65 .mu.g/kg or less, 60
.mu.g/kg or less, 55 .mu.g/kg or less, 50 .mu.g/kg or less, 45
.mu.g/kg or less, 40 .mu.g/kg or less, 35 .mu.g/kg or less, 30
.mu.g/kg or less, 25 .mu.g/kg or less, 20 .mu.g/kg or less, 15
.mu.g/kg or less, 10 .mu.g/kg or less, 5 .mu.g/kg or less, 2.5
.mu.g/kg or less, 2 .mu.g/kg or less, 1.5 .mu.g/kg or less, 1
.mu.g/kg or less, 0.5 .mu.g/kg or less, or 0.5 .mu.g/kg or less of
a patient's body weight.
[0311] Unit dose of the scaffolds of the invention may be 0.1 mg to
20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to
8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20
mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25
mg to 7 m g, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg
to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg,
1 mg to 5 mg, or 1 mg to 2.5 mg.
[0312] The dosage of the scaffolds of the invention may achieve a
serum titer of at least 0.1 .mu.g/ml, at least 0.5 .mu.g/ml, at
least 1 .mu.g/ml, at least 2 .mu.g/ml, at least 5 .mu.g/ml, at
least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15 .mu.g/ml, at
least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50 .mu.g/ml, at
least 100 .mu.g/ml, at least 125 .mu.g/ml, at least 150 .mu.g/ml,
at least 175 .mu.g/ml, at least 200 .mu.g/ml, at least 225
.mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml, at least
300 .mu.g/ml, at least 325 .mu.g/ml, at least 350 .mu.g/ml, at
least 375 .mu.g/ml, or at least 400 .mu.g/ml in a subject.
Alternatively, the dosage of the scaffolds of the invention may
achieve a serum titer of at least 0.1 .mu.g/ml, at least 0.5
.mu.g/ml, at least 1 .mu.g/ml, at least, 2 .mu.g/ml, at least 5
.mu.g/ml, at least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15
.mu.g/ml, at least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50
.mu.g/ml, at least 100 .mu.g/ml, at least 125 .mu.g/ml, at least
150 .mu.g/ml, at least 175 .mu.g/ml, at least 200 .mu.g/ml, at
least 225 .mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml,
at least 300 .mu.g/ml, at least 325 .mu.g/ml, at least 350
.mu.g/ml, at least 375 .mu.g/ml, or at least 400 .mu.g/ml in the
subject.
[0313] Doses of scaffolds of the invention may be repeated and the
administrations may be separated by at least 1 day, 2 days, 3 days,
5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3
months, or at least 6 months.
[0314] An effective amount for a particular patient may vary
depending on factors such as the condition being treated, the
overall health of the patient, the method route and dose of
administration and the severity of side affects (see, e.g.,
Maynard, et al. (1996) A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good
Laboratory and Good Clinical Practice, Urch Publ., London, UK).
[0315] The route of administration may be by, e.g., topical or
cutaneous application, injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intracerebrospinal, intralesional, or by sustained
release systems or an implant (see, e.g., Sidman et al. (1983)
Biopolymers 22:547-556; Langer, et al. (1981) J. Biomed. Mater.
Res. 15:167-277; Langer (1982) Chem. Tech. 12:98-105; Epstein, et
al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al.
(1980) Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos.
6,350,466 and 6,316,024). Where necessary, the composition may also
include a solubilizing agent and a local anesthetic such as
lidocaine to ease pain at the site of the injection. In addition,
pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309,
5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT
Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO
98/31346, and WO 99/66903, each of which is incorporated herein by
reference their entirety. In one embodiment, an antibody,
combination therapy, or a composition of the invention is
administered using Alkermes AIR.TM. pulmonary drug delivery
technology (Alkermes, Inc., Cambridge, Mass.).
[0316] A composition of the present invention may also be
administered via one or more routes of administration using one or
more of a variety of methods known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
Selected routes of administration for scaffolds of the invention
include intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal or other parenteral routes of administration,
for example by injection or infusion. Parenteral administration may
represent modes of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion. Alternatively, a
composition of the invention can be administered via a
non-parenteral route, such as a topical, epidermal or mucosal route
of administration, for example, intranasally, orally, vaginally,
rectally, sublingually or topically.
[0317] If the scaffolds of the invention are administered in a
controlled release or sustained release system, a pump may be used
to achieve controlled or sustained release (see Langer, supra;
Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al.,
1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.
321:574). Polymeric materials can be used to achieve controlled or
sustained release of the therapies of the invention (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253. Examples of polymers
used in sustained release formulations include, but are not limited
to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),
poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one
embodiment, the polymer used in a sustained release formulation is
inert, free of leachable impurities, stable on storage, sterile,
and biodegradable. A controlled or sustained release system can be
placed in proximity of the prophylactic or therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)).
[0318] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more scaffolds of the invention.
See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548,
PCT publication WO 96/20698, Ning et al., 1996, "Intratumoral
Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a
Sustained-Release Gel," Radiotherapy & Oncology 39:179-189,
Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology 50:372-397, Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854, and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of
which is incorporated herein by reference in their entirety.
[0319] If the scaffold of the invention is administered topically,
it can be formulated in the form of an ointment, cream, transdermal
patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or
other form well-known to one of skill in the art. See, e.g.,
Remington's Pharmaceutical Sciences and Introduction to
Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.
(1995). For non-sprayable topical dosage forms, viscous to
semi-solid or solid forms comprising a carrier or one or more
excipients compatible with topical application and having a dynamic
viscosity, in some instances, greater than water are typically
employed. Suitable formulations include, without limitation,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and the like, which are, if desired, sterilized
or mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, in some instances, in combination with a
solid or liquid inert carrier, is packaged in a mixture with a
pressurized volatile (e.g., a gaseous propellant, such as freon) or
in a squeeze bottle. Moisturizers or humectants can also be added
to pharmaceutical compositions and dosage forms if desired.
Examples of such additional ingredients are well-known in the
art.
[0320] If the scaffolds of the invention are administered
intranasally, it can be formulated in an aerosol form, spray, mist
or in the form of drops. In particular, prophylactic or therapeutic
agents for use according to the present invention can be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebuliser, with the use of a suitable
propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0321] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are well known in the art (see,
e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill,
New York, N.Y.; Poole and Peterson (eds.) (2001)
Pharmacotherapeutics for Advanced Practice:A Practical Approach,
Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo
(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,
Williams & Wilkins, Phila., Pa.). An effective amount of
therapeutic may decrease the symptoms by at least 10%; by at least
20%; at least about 30%; at least 40%, or at least 50%.
[0322] Additional therapies (e.g., prophylactic or therapeutic
agents), which can be administered in combination with the
scaffolds of the invention may be administered less than 5 minutes
apart, less than 30 minutes apart, 1 hour apart, at about 1 hour
apart, at about 1 to about 2 hours apart, at about 2 hours to about
3 hours apart, at about 3 hours to about 4 hours apart, at about 4
hours to about 5 hours apart, at about 5 hours to about 6 hours
apart, at about 6 hours to about 7 hours apart, at about 7 hours to
about 8 hours apart, at about 8 hours to about 9 hours apart, at
about 9 hours to about 10 hours apart, at about 10 hours to about
11 hours apart, at about 11 hours to about 12 hours apart, at about
12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to
36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours
apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72
hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to
120 hours apart from the scaffolds of the invention. The two or
more therapies may be administered within one same patient
visit.
[0323] The scaffolds of the invention and the other therapies may
be cyclically administered. Cycling therapy involves the
administration of a first therapy (e.g., a first prophylactic or
therapeutic agent) for a period of time, followed by the
administration of a second therapy (e.g., a second prophylactic or
therapeutic agent) for a period of time, optionally, followed by
the administration of a third therapy (e.g., prophylactic or
therapeutic agent) for a period of time and so forth, and repeating
this sequential administration, i.e., the cycle in order to reduce
the development of resistance to one of the therapies, to avoid or
reduce the side effects of one of the therapies, and/or to improve
the efficacy of the therapies.
[0324] In certain embodiments, the scaffolds of the invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods
4:273.
[0325] The invention provides protocols for the administration of
pharmaceutical composition comprising scaffolds of the invention
alone or in combination with other therapies to a subject in need
thereof. The therapies (e.g., prophylactic or therapeutic agents)
of the combination therapies of the present invention can be
administered concomitantly or sequentially to a subject. The
therapy (e.g., prophylactic or therapeutic agents) of the
combination therapies of the present invention can also be
cyclically administered. Cycling therapy involves the
administration of a first therapy (e.g., a first prophylactic or
therapeutic agent) for a period of time, followed by the
administration of a second therapy (e.g., a second prophylactic or
therapeutic agent) for a period of time and repeating this
sequential administration, i.e., the cycle, in order to reduce the
development of resistance to one of the therapies (e.g., agents) to
avoid or reduce the side effects of one of the therapies (e.g.,
agents), and/or to improve, the efficacy of the therapies.
[0326] The therapies (e.g., prophylactic or therapeutic agents) of
the combination therapies of the invention can be administered to a
subject concurrently. The term "concurrently" is not limited to the
administration of therapies (e.g., prophylactic or therapeutic
agents) at exactly the same time, but rather it is meant that a
pharmaceutical composition comprising scaffolds of the invention
are administered to a subject in a sequence and within a time
interval such that the scaffolds of the invention can act together
with the other therapy(ies) to provide an increased benefit than if
they were administered otherwise. For example, each therapy may be
administered to a subject at the same time or sequentially in any
order at different points in time; however, if not administered at
the same time, they should be administered sufficiently close in
time so as to provide the desired therapeutic or prophylactic
effect. Each therapy can be administered to a subject separately,
in any appropriate form and by any suitable route. In various
embodiments, the therapies (e.g., prophylactic or therapeutic
agents) are administered to a subject less than 15 minutes, less
than 30 minutes, less than 1 hour apart, at about 1 hour apart, at
about 1 hour to about 2 hours apart, at about 2 hours to about 3
hours apart, at about 3 hours to about 4 hours apart, at about 4
hours to about 5 hours apart, at about 5 hours to about 6 hours
apart, at about 6 hours to about 7 hours apart, at about 7 hours to
about 8 hours apart, at about 8 hours to about 9 hours apart, at
about 9 hours to about 10 hours apart, at about 10 hours to about
11 hours apart, at about 11 hours to about 12 hours apart, 24 hours
apart, 48 hours apart, 72 hours apart, or 1 week apart. In other
embodiments, two or more therapies (e.g., prophylactic or
therapeutic agents) are administered to a within the same patient
visit.
[0327] The prophylactic or therapeutic agents of the combination
therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, the prophylactic or
therapeutic agents of the combination therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
The prophylactic or therapeutic agents may be administered to a
subject by the same or different routes of administration.
6.16 Methods of Using Scaffolds
[0328] The scaffolds of the present invention have in vitro and in
vivo diagnostic and therapeutic utilities. For example, these
molecules can be administered to cells in culture, e.g. in vitro or
ex vivo, or in a subject, e.g., in vivo, to treat, prevent or
diagnose a variety of disorders.
[0329] The invention also provides methods of using the scaffolds
of the invention. The present invention also encompasses the use of
the scaffolds of the invention for the prevention, diagnosis,
management, treatment or amelioration of one or more symptoms
associated with diseases, disorders of diseases or disorders,
including but not limited to cancer, inflammatory and autoimmune
diseases, infectious diseases either alone or in combination with
other therapies. The invention also encompasses the use of the
scaffolds of the invention conjugated or fused to a moiety (e.g.,
therapeutic agent or drug) for prevention, management, treatment or
amelioration of one or more symptoms associated with diseases,
disorders or infections, including but not limited to cancer,
inflammatory and autoimmune diseases, infectious diseases either
alone or in combination with other therapies.
[0330] Also, many cell surface receptors activate or deactivate as
a consequence of crosslinking of subunits. The proteins of the
invention may be used to stimulate or inhibit a response in a
target cell by crosslinking of cell surface receptors. In another
embodiment, the scaffolds of the invention of the invention may be
used to block the interaction of multiple cell surface receptors
with antigens. In another embodiment, the scaffolds of the
invention may be used to strengthen the interaction of multiple
cell surface receptors with antigens. In another embodiment, it may
be possible to crosslink homo- or heterodimers of a cell surface
receptor using the scaffolds of the invention containing binding
domains that share specificity for the same antigen, or bind two
different antigens. In another embodiment, the proteins of the
invention could be used to deliver a ligand, or ligand analogue to
a specific cell surface receptor.
[0331] The invention also provides methods of targeting epitopes
not easily accomplished with traditional antibodies. For example,
in one embodiment, the scaffolds and of the invention may be used
to first target an adjacent antigen and while binding, another
binding domain may engage the cryptic antigen.
[0332] The invention also provides methods of using the scaffolds
to bring together distinct cell types. In one embodiment, the
proteins of the invention may bind a target cell with one binding
domain and recruit another cell via another binding domain. In
another embodiment, the first cell may be a cancer cell and the
second cell is an immune effector cell such as an NK cell. In
another embodiment, the scaffolds of the invention may be used to
strengthen the interaction between two distinct cells, such as an
antigen presenting cell and a T cell to possibly boost the immune
response.
[0333] The invention also provides methods of using the scaffolds
proteins to ameliorate, treat, or prevent cancer or symptoms
thereof. In one embodiment, methods of the invention are useful in
the treatment of cancers of the head, neck, eye, mouth, throat,
esophagus, chest, skin, bone, lung, colon, rectum, colorectal,
stomach, spleen, kidney, skeletal muscle, subcutaneous tissue,
metastatic melanoma, endometrial, prostate, breast, ovaries,
testicles, thyroid, blood, lymph nodes, kidney, liver, pancreas,
brain, or central nervous system.
[0334] The invention also provides methods of using the scaffolds
to deplete a cell population. In one embodiment, methods of the
invention are useful in the depletion of the following cell types:
eosinophil, basophil, neutrophil, T cell, B cell, mast cell,
monocytes and tumor cell.
6.17 TRAIL-R2 Specific Scaffolds
[0335] The TRAIL-R2 protein is encoded by a member of the
TNF-receptor superfamily gene, and contains an intracellular death
domain. In some instances, it may also be known as TNFRSF10B;
CD262, DR5, KILLER<KILLER/DR5, TRAILR2, TRICK2, TRICK2A,
TRICK2B, TRICKB, or ZTNFR9. This receptor can be activated by tumor
necrosis factor-related apoptosis inducing ligand
(TNFSF10/TRAIL/APO-2L), and transduces an apoptotic signal.
Further, TRAIL-R2 induced apoptosis involves caspases and the
intracelular adapter molecule FADD/MORT1 (Walczak et al. EMBOJ,
(1997), 16, 5386-97).
[0336] In some embodiments, the invention also provides scaffolds
that specifically bind to TRAIL-R2. In specific embodiments,
scaffolds of the invention specifically bind to human TRAIL-R2. In
other specific embodiments, scaffolds of the invention bind to
TRAIL-R2 homologs from mouse, chicken, Rheses, cynomolgus, rat, or
rabbit. In some embodiments, scaffolds of the invention bind to an
exposed epitope of TRAIL-R2. Such embodiments include TRAIL-R2
endogenously expressed on cells and/or cells transfected to
ectopically express the receptor. In other embodiments, scaffolds
of the invention recognize epitopes displayed on a monomeric
TRAIL-R2. In other embodiments, scaffolds of the invention
recognize epitopes displayed on a homodimeric form of TRAIL-R2. In
yet other embodiments, scaffolds of the invention bind monomeric
TRAIL-R2 and facilitate dimerization or oligomerization of 2 or
more TRAIL-R2 molecules (for example, but not limited to multimeric
scaffolds). In yet other embodiments, scaffolds of the invention
reduce or inhibit interaction of TRAIL-R2 with TRAIL ligand. In
other embodiments, scaffolds of the invention mimic the interaction
of TRAIL ligand with TRAIL-R2. In further embodiments, scaffolds of
the invention agonize cellular signaling by TRAIL-R2.
[0337] The invention also provides methods of modulating TRAIL-R2
activity using the scaffolds described herein. In some embodiments,
methods of the invention comprise contacting a cell expressing
TRAIL-R2 with TRAIL-R2 specific scaffolds and blocking interaction
with TRAIL ligand. In other embodiments, methods of the invention
comprise contacting a cell expressing TRAIL-R2 with a TRAIL-R2
specific scaffold and mimicking the interaction of TRAIL ligand
with TRAIL-R2. In other embodiments, methods of the invention
comprise agonizing TRAIL-R2 by contacting with a TRAIL-R2 specific
scaffold. In other embodiments, methods of the invention comprise
dimerizing or oligomerize TRAIL-R2 by contacting a monomer of
TRAIL-R2 expressed on cells with a TRAIL-R2 specific scaffold and
facilitating dimerization or oligomerization. In further
embodiments, dimerization of TRAIL-R2 may be achieved through the
use of, for example, but not limited to, multimeric scaffolds,
scaffolds that mimic TRAIL-R2 dimers, scaffolds that stabilize
TRAIL-R2 dimer formation, scaffolds that destabilize TRAIL-R2
monomers or scaffolds that only recognize TRAIL-R2 dimers displayed
on cells.
[0338] In other embodiments, dimerization or oligomerization of
TRAIL-R2 may be achieved through the use of monomeric scaffolds
coupled with a scaffold dimerization or oligomerization agent. Such
scaffolds dimerization or oligomerization agents may include, for
example, but not limited to, an anti-scaffold antibody, use of
scaffolds with epitope tags coupled with antibodies to epitope tag,
or the incorporation of various protein dimerization or
oligomerization motifs described herein and known in the art. In a
further embodiment, TRAIL-R2 dimers or oligomers may be induced by
the administration of monomeric scaffolds followed by the
administration of a scaffold dimerization or oligomerization
agent.
[0339] In some embodiments, methods of the invention comprise the
administration of a TRAIL-R2 specific scaffold that reduces cell
viability as measured by routine assays known in the art. In
further embodiments, the reduction in cell viability is activation
of apoptosis as measured by known assays in the art. In other
embodiments, reduction in cell viability is the inhibition of cell
division as measured by art accepted methods. In some embodiments,
cell viability is reduced by at least 10%, at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, or more as compared to cell viability in
the absence of treatment. In some embodiments, cell viability is
measured using the procedure outlined in Example 15 and/or 17
herein or other known methods in the art.
[0340] In some embodiments, TRAIL-R2 binding scaffolds of the
invention agonize TRAIL-R2 with similar activity as the ligand for
TRAIL-R2, known as TRAIL (Apo-2 ligand). In other embodiments,
TRAIL-R2 binding scaffolds of the invention are capable of
sufficiently activating TRAIL-R2 to result in the activation of one
or more intracellular signaling pathways, including the activation
of caspase 3, caspase 8, caspase 10, or FADD. In other embodiments,
TRAIL-R2 binding scaffolds of the invention activate apoptosis in
at least one cancer cell type. In further embodiments, TRAIL-R2
binding scaffolds of the invention demonstrate an enhanced
activation of apoptosis in at least one cell type as compared to
TRAIL. In other embodiments, the TRAIL-R2 binding scaffolds of the
invention may bind or compete with binding for the same epitope on
TRAIL-R2 as TRAIL (ligand). In such embodiments, the TRAIL-R2
binding scaffolds are capable of blocking of inhibiting the
interaction of TRAIL-R2 with TRAIL by at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, or more which may be
determined in an in vitro competitive assay using the soluble TRAIL
ligand (such as the 114-281 fragment of TRAIL ligand),
crystallographic studies, or other known in vivo or in vitro
studies.
6.17.1 Methods of Using TRAIL-R2 Binders in Therapy
[0341] TRAIL-R2 is known to mediate apoptosis signaling. Although
several types of normal cells express TRAIL-R2, apoptosis signaling
through this receptor appears to be restricted primarily to tumor
cells, which become more susceptible to death receptor-mediated
apoptosis in the context of their transformation by oncogenes such
as Myc or Ras (Wang et al., Cancer Cell 5:501-12 (2004); Nesterov
et al., Cancer Res. 64:3922-7 (2004)). TRAIL-R2 is frequently
expressed by human cancer cell lines as well as primary tumors.
[0342] The TRAIL-R2 specific scaffolds of the invention may be
useful in the prevention, treatment, maintenance or amelioration of
cancer. In some embodiments, cancer may involve cancer cells that
express TRAIL-R2. In other embodiments, cancer cells overexpress
TRAIL-R2 as compared to non-cancerous cells. In some embodiments,
the cancer is, for example, carcinoma, lymphoma, blastoma, sarcoma,
or leukemia. In other embodiments, cancer may include squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer (NSCLC),
non-Hodgkin's lymphoma, blastoma, gastrointestinal cancer, renal
cancer, ovarian cancer, liver cancer, stomach cancer, bladder
cancer, hepatoma, breast cancer, colon cancer, colorectal cancer,
pancreatic cancer, endometrial carcinoma, salivary gland carcinoma,
kidney cancer, liver cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, head and neck cancer, lung
cancer, adenocarcinoma, renal cell carcinoma, hepatocellular
carcinoma, or other cancers described herein.
[0343] In some embodiments, TRAIL-R2 specific scaffolds of the
invention are administered to a subject in need of treatment (i.e.
a patient with cancer). In such embodiments, a sterile,
pyrogen-free composition comprising a TRAIL-R2 specific scaffold is
administered to a subject in need thereof. The efficiency of
treatment may be measured using a variety of in vitro and in vivo
assays well known in the art, such as, but not limited to apoptotic
activity, using caspase activation of Annexin V binding, as well as
a reduction in tumor burden or volume.
[0344] In other embodiments, TRAIL-R2 specific scaffolds of the
invention are useful for the diagnosis and detection of cancer or
other TRAIL-R2 associated diseases. In such embodiments, TRAIL-R2
specific scaffolds of the invention are linked to a detection
agent, such as, but not limited to a radioisotope, fluorescent or
chemiluminescent label. Such linked binders are useful in methods
that detect or diagnose cancer or TRAIL-R2 associated diseases in a
subject, or a sample taken from said subject. In addition, TRAIL-R2
specific scaffolds are useful in the diagnosis and treatment of
other TRAIL-R2 associated pathological conditions, such as
immune-related diseases in mammals, including humans.
6.17.2 Specific TRAIL-R2 Binding Sequences
[0345] In an effort to identify TRAIL-R2 specific scaffolds, a
two-loop library and a three loop library were screened. A number
of clones were identified as specifically binding to TRAIL-R2.
[0346] In some embodiments TRAIL-R2 specific scaffolds of the
invention comprise at least one, at least two, at least three, at
least four, at least five, or at least six loop sequences that bind
TRAIL-R2. In some embodiments, TRAIL-R2 specific scaffolds comprise
at least one, at least two, at least three, at least four, at least
five, or at least six loop sequences of TRAIL-R2 binding scaffold
clones selected from 2F4, 5B10, 10D9, 6F11, 8B3, 5E5, 2H6, 7G11, or
6C7. In other embodiments, TRAIL-R2 specific scaffolds comprise at
least one loop sequence selected from SEQ ID NOs:126-143. In other
embodiments, TRAIL-R2 specific scaffolds comprise at least one BC
loop sequence selected from SEQ ID NOs: 126, 128, 130, 132, 134,
136, 138, 140, or 142. In other embodiments, TRAIL-R2 specific
scaffolds comprise at least one FG loop sequence selected from SEQ
ID NOs: 127, 129, 131, 133, 135, 137, 139, 141, or 143. In other
embodiments, TRAIL-R2 specific scaffolds comprise a BC loop
sequence selected from SEQ ID NOs:126, 128, 130, 132, 134, 136,
138, 140, or 142; and an FG loop sequence selected from SEQ ID
NOs:127, 129, 131, 133, 135, 137, 139, 141, or 143. In a specific
embodiment, TRAIL-R2 specific scaffolds comprise a BC loop sequence
of SEQ ID NO:126 and an FG loop sequence of 127. In a specific
embodiment, TRAIL-R2 specific scaffolds comprise a BC loop sequence
of SEQ ID NO: 128 and a FG loop sequence of SEQ ID NO: 129. In a
specific embodiment, TRAIL-R2 specific scaffolds comprise a BC loop
sequence of SEQ ID NO:130 and a FG loop sequence of SEQ ID NO:131.
In a specific embodiment, TRAIL-R2 specific scaffolds comprise a BC
loop sequence of SEQ ID NO:132 and a FG loop sequence of SEQ ID
NO:133. In a specific embodiment, TRAIL-R2 specific scaffolds
comprise a BC loop sequence of SEQ ID NO: 134 and a FG loop
sequence of SEQ ID NO:135. In a specific embodiment, TRAIL-R2
specific scaffolds comprise a BC loop sequence of SEQ ID NO:136 and
a FG loop sequence of SEQ ID NO:137. In another specific
embodiment, TRAIL-R2 specific scaffolds comprise a BC loop sequence
of SEQ ID NO:140 and an FC loop sequence of SEQ ID NO:141. In yet
another specific embodiment, TRAIL-R2 specific scaffolds comprise a
BC loop sequence of SEQ ID NO:138 and an FC loop sequence of SEQ ID
NO:139.
[0347] In other embodiments, the invention may also comprise
scaffolds which compete for binding with scaffolds that
specifically bind TRAIL-R2, said TRAIL-R2 binders, selected from
the group consisting of 2F4, 5B10, 10D9, 6F11, 8B3, 5E5, 2Hb, 7G11,
or 6C7. In other embodiments, the invention may also comprise
scaffolds which compete for binding with scaffolds that
specifically bind TRAIL-R2, said TRAIL-R2 binders comprising one BC
loop sequence selected from SEQ ID NOs: 126, 128, 130, 132, 134,
136, 138, 140, or 142; and one FG loop sequence selected from SEQ
ID NOs: 127, 129, 131, 133, 135, 137, 139, 141, or 143. Competition
assays may be performed as presented herein in Examples 11 and/or
14, or by other assays known in the art.
[0348] In other embodiments, TRAIL-R2 specific scaffolds comprise
at least one, at least two, at least three, at least four, at least
five, or at least six loop sequences of TRAIL-R2 binding scaffold
clones selected from 1E03, 2B04, 1C12, 1A03, 1C10, 1B12, 2G03, 2D3,
1C06, 2F08, 1B04, 3B11, 1D8, 2A12, 1E05, 2F02, 1H05, 2A11, or 1G11.
In other embodiments, TRAIL-R2 specific scaffolds comprise at least
one loop from SEQ ID NOs:144-200. In other embodiments, TRAIL-R2
specific scaffolds comprise at least one BC loop sequence selected
from SEQ ID NO:144, 147, 150, 153, 156, 159, 162, 165, 168, 171,
174, 177, 180, 183, 186, 189, 192, 195, or 198. In other
embodiments, TRAIL-R2 specific scaffolds comprise at least one DE
loop sequence selected from SEQ ID NO:145, 148, 151, 154, 157, 160,
163, 166, 169, 172, 175, 178, 181, 194, 187, 190, 193, 196, or 199.
In other embodiments, TRAIL-R2 specific scaffolds comprise at least
one FG loop sequence selected from SEQ ID NO:146, 149, 152, 155,
158, 161, 164, 167, 170, 173, 179, 182, 185, 188, 191, 194, 197, or
200. In further embodiments, TRAIL-R2 specific scaffolds comprise
at least one BC loop sequence selected from SEQ ID NO:144, 147,
150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186,
189, 192, 195, or 198; at least one DE loop sequence selected from
SEQ ID NO:145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175,
178, 181, 194, 187, 190, 193, 196, or 199; and at least one FG loop
sequence selected from SEQ ID NO:146, 149, 152, 155, 158, 161, 164,
167, 170, 173, 179, 182, 185, 188, 191, 194, 197, or 200.
[0349] In a specific embodiment, TRAIL-R2 specific scaffolds
comprise a BC loop sequence of SEQ ID NO:144, a DE loop sequence of
SEQ ID NO:145, and an FG loop sequence of SEQ ID NO:146. In a
specific embodiment, TRAIL-R2 specific scaffolds comprise a BC loop
sequence of SEQ ID NO:147, a DE loop sequence of SEQ ID NO:148, and
an FG loop sequence of SEQ ID NO:149. In a specific embodiment,
TRAIL-R2 specific scaffolds comprise a BC loop sequence of SEQ ID
NO:150, a DE loop sequence of SEQ ID NO:151, and an FG loop
sequence of SEQ ID NO:152. In a specific embodiment, TRAIL-R2
specific scaffolds comprise a BC loop sequence of SEQ ID NO:153, a
DE loop sequence of SEQ ID NO:154, and an FG loop sequence of SEQ
ID NO:155. In a specific embodiment, TRAIL-R2 specific scaffolds
comprise a BC loop sequence of SEQ ID NO:165, a DE loop sequence of
SEQ ID NO:166, and an FG loop sequence of SEQ ID NO:167. In a
specific embodiment, TRAIL-R2 specific scaffolds comprise a BC loop
sequence of SEQ ID NO:198, a DE loop sequence of SEQ ID NO:199, and
an FG loop sequence of SEQ ID NO:200.
[0350] In other embodiments, the invention may also comprise
scaffolds which compete for binding with scaffolds that
specifically bind TRAIL-R2, said TRAIL-R2 binders, selected from
the group consisting of 1E03, 2B04, 1C12, 1A03, 1C10, 1B12, 2G03,
2D3, 1C06, 2F08, 1B04, 3B11, 1D8, 2A12, 1E05, 2F02, 1H05, 2A11, or
1G11. In other embodiments, the invention may also comprise
scaffolds which compete for binding with scaffolds that
specifically bind TRAIL-R2, said TRAIL-R2 binders comprising one BC
loop sequence selected from SEQ ID NO:144, 147, 150, 153, 156, 159,
162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, or 198;
at least one DE loop sequence selected from SEQ ID NO:145, 148,
151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 194, 187,
190, 193, 196, or 199; and at least one FG loop sequence selected
from SEQ ID NO:146, 149, 152, 155, 158, 161, 164, 167, 170, 173,
179, 182, 185, 188, 191, 194, 197, or 200. Competition assays may
be performed as presented herein in Examples 11 and/or 14, or by
other assays known in the art.
6.18 Methods of Use for Scaffolds
[0351] The invention also provides methods of using scaffolds to
inactivate, inhibit, or deplete cytokines. In one embodiment,
methods of the invention are useful in the inactivation,
inhibition, or depletion of at least one of the following
cytokines: TNF-.alpha., TGF-.beta., C5a, fMLP, Interferon alpha
(including subtypes 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17
and 21), Interferon beta, Interferon omega, Interferon gamma,
interleukins CCL 1-28, CXCL 1-17, and CX3CL1.
[0352] The invention also provides methods of using the scaffolds
to inactivate various infections agents such as viruses, fungi,
eukaryotic microbes, and bacteria. In some embodiments the
scaffolds of the invention may be used to inactivate RSV, hMPV,
PIV, or influenza viruses. In other embodiments, the scaffolds of
the invention may be used to inactivate fungal pathogens, such as,
but not limited to members of Naegleria, Aspergillus, Blastomyces,
Histoplasma, Candida or Tinea genera. In other embodiments, the
scaffolds of the invention may be used to inactivate eukaryotic
microbes, such as, but not limited to members of Giardia,
Toxoplasma, Plasmodium, Trypanosoma, and Entamoeba genera. In other
embodiments, the scaffolds of the invention may be used to
inactivate bacterial pathogens, such as but not limited to members
of Staphylococcus, Streptococcus, Pseudomonas, Clostridium,
Borrelia, Vibro and Neiserria genera.
[0353] The invention also provides methods of using scaffolds
proteins as diagnostic reagents. The proteins of the invention may
be useful in kits or reagents where different antigens need to be
efficiently captured concurrently.
[0354] The proteins of the invention and compositions comprising
the same are useful for many purposes, for example, as therapeutics
against a wide range of chronic and acute diseases and disorders
including, but not limited to, cancer. Examples of cancers that can
be prevented, managed, treated or ameliorated in accordance with
the methods of the invention include, but are not limited to,
cancer of the head, neck, eye, mouth, throat, esophagus, chest,
bone, lung, colon, rectum, stomach, prostate, breast, ovaries,
kidney, liver, pancreas, and brain. Additional cancers include, but
are not limited to, the following: leukemias such as but not
limited to, acute leukemia, acute lymphocytic leukemia, acute
myelocytic leukemias such as myeloblastic, promyelocytic,
myelomonocytic, monocytic, erythroleukemia leukemias and
myclodysplastic syndrome, chronic leukemias such as but not limited
to, chronic myclocytic (granulocytic) leukemia, chronic lymphocytic
leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as
but not limited to Hodgkin's disease, non-Hodgkin's disease;
multiple myelomas such as but not limited to smoldering multiple
mycloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell
leukemia, solitary plasmacytoma and extramedullary plasmacytoma;
Waldenstrom's macroglobulinemia; monoclonal gammopathy of
undetermined significance; benign monoclonal gammopathy; heavy
chain disease; bone cancer and connective tissue sarcomas such as
but not limited to bone sarcoma, myeloma bone disease, multiple
myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease
of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordoma, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, and synovial
sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, and
primary brain lymphoma; breast cancer including but not limited to
adenocarcinoma, lobular (small cell) carcinoma, intraductal
carcinoma, medullary breast cancer, mucinous breast cancer, tubular
breast cancer, papillary breast cancer, Paget's disease (including
juvenile Paget's disease) and inflammatory breast cancer; adrenal
cancer such as but not limited to pheochromocytom and
adrenocortical carcinoma; thyroid cancer such as but not limited to
papillary or follicular thyroid cancer, medullary thyroid cancer
and anaplastic thyroid cancer; pancreatic cancer such as but not
limited to, insulinoma, gastrinoma, glucagonoma, vipoma,
somatostatin-secreting tumor, and carcinoid or islet cell tumor;
pituitary cancers such as but limited to Cushing's disease,
prolactin-secreting tumor, acromegaly, and diabetes insipius; eye
cancers such as but not limited to ocular melanoma such as iris
melanoma, choroidal melanoma, and cilliary body melanoma, and
retinoblastoma; vaginal cancers such as squamous cell carcinoma,
adenocarcinoma, and melanoma; vulvar cancer such as squamous cell
carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,
and Paget's disease; cervical cancers such as but not limited to,
squamous cell carcinoma, and adenocarcinoma; uterine cancers such
as but not limited to endometrial carcinoma and uterine sarcoma;
ovarian cancers such as but not limited to, ovarian epithelial
carcinoma, borderline tumor, germ cell tumor, and stromal tumor;
esophageal cancers such as but not limited to, squamous cancer,
adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma,
adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous
carcinoma, and oat cell (small cell) carcinoma; stomach cancers
such as but not limited to, adenocarcinoma, fungaling (polypoid),
ulcerating, superficial spreading, diffusely spreading, malignant
lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon
cancers; rectal cancers; liver cancers such as but not limited to
hepatocellular carcinoma and hepatoblastoma, gallbladder cancers
such as adenocarcinoma; cholangiocarcinomas such as but not limited
to pappillary, nodular, and diffuse; lung cancers such as non-small
cell lung cancer, squamous cell carcinoma (epidermoid carcinoma),
adenocarcinoma, large-cell carcinoma and small-cell lung cancer;
testicular cancers such as but not limited to germinal tumor,
seminoma, anaplastic, classic (typical), spermatocytic,
nonseminoma, embryonal carcinoma, teratoma carcinoma,
choriocarcinoma (yolk-sac tumor), prostate cancers such as but not
limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma;
penal cancers; oral cancers such as but not limited to squamous
cell carcinoma; basal cancers; salivary gland cancers such as but
not limited to adenocarcinoma, mucoepidermoid carcinoma, and
adenoidcystic carcinoma; pharynx cancers such as but not limited to
squamous cell cancer, and verrucous; skin cancers such as but not
limited to, basal cell carcinoma, squamous cell carcinoma and
melanoma, superficial spreading melanoma, nodular melanoma, lentigo
malignant melanoma, acral lentiginous melanoma; kidney cancers such
as but not limited to renal cell cancer, adenocarcinoma,
hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis
and/or ureter); Wilms' tumor; bladder cancers such as but not
limited to transitional cell carcinoma, squamous cell cancer,
adenocarcinoma, carcinosarcoma. In addition, cancers include
myxosarcoma, osteogenic sarcoma, endotheliosarcoma,
lymphangioendotheliosarcoma, mesothelioma, synovioma,
hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,
bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma and papillary adenocarcinomas (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997,
Informed Decisions: The Complete Book of Cancer Diagnosis,
Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A.,
inc., United States of America). It is also contemplated that
cancers caused by aberrations in apoptosis can also be treated by
the methods and compositions of the invention. Such cancers may
include, but not be limited to, follicular lymphomas, carcinomas
with p53 mutations, hormone dependent tumors of the breast,
prostate and ovary, and precancerous lesions such as familial
adenomatous polyposis, and myelodysplastic syndromes.
[0355] The proteins of the invention and compositions comprising
the same are useful for many purposes, for example, as therapeutics
against a wide range of chronic and acute diseases and disorders
including, but not limited to, autoimmune and/or inflammatory
diseases. The compositions and methods of the invention described
herein are useful for the prevention or treatment of autoimmune
disorders and/or inflammatory disorders. Examples of autoimmune
and/or inflammatory disorders include, but are not limited to,
alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,
autoimmune Addison's disease, autoimmune diseases of the adrenal
gland, autoimmune hemolytic anemia, autoimmune hepatitis,
autoimmune oophoritis and orchitis, Sjogren's syndrome, psoriasis,
atherosclerosis, diabetic and other retinopathies, retrolental
fibroplasia, age-related macular degeneration, neovascular
glaucoma, hemangiomas, thyroid hyperplasias (including Grave's
disease), corneal and other tissue transplantation, and chronic
inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's
disease, reperfusion injury, septicemia, endotoxic shock, cystic
fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic
arthritis), anaphylactic shock, organ ischemia, reperfusion injury,
spinal cord injury and allograft rejection. autoimmune
thrombocytopenia, Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune
dysfunction syndrome (CFIDS), chronic inflammatory demyelinating
polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid,
CREST syndrome, cold agglutinin disease, Crohn's disease, discoid
lupus, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary
fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA
neuropathy, juvenile arthritis, lichen planus, lupus erythematosus,
Meniere's disease, mixed connective tissue disease, multiple
sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia
gravis, pemphigus vulgaris, pernicious anemia, polyarteritis
nodosa, polychrondritis, polyglandular syndromes, polymyalgia
rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid
arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man
syndrome, systemic lupus erythematosus, lupus erythematosus,
takayasu arteritis, temporal arteristis/giant cell arteritis,
ulcerative colitis, uveitis, vasculitides such as dermatitis
herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
Examples of inflammatory disorders include, but are not limited to,
asthma, encephilitis, inflammatory bowel disease, chronic
obstructive pulmonary disease (COPD), allergic disorders, septic
shock, pulmonary fibrosis, undifferentitated spondyloarthropathy,
undifferentiated arthropathy, arthritis, inflammatory osteolysis,
and chronic inflammation resulting from chronic viral or bacteria
infections. The compositions and methods of the invention can be
used with one or more conventional therapies that are used to
prevent, manage or treat the above diseases.
[0356] The proteins of the invention and compositions comprising
the same are useful for many purposes, for example, as therapeutics
against a wide range of chronic and acute diseases and disorders
including, but not limited to, infectious disease, including viral,
bacterial and fungal diseases. Examples of viral pathogens include
but are not limited to: adenovirdiae (e.g., mastadenovirus and
aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes
simplex virus 2, herpes simplex virus 5, and herpes simplex virus
6), leviviridae (e.g., levivirus, enterobacteria phase MS2,
allolevirus), poxyiridae (e.g., chordopoxyirinae, parapoxvirus,
avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus,
molluscipoxvirus, and entomopoxyirinae), papovaviridae (e.g.,
polyomavirus and papillomavirus), paramyxoviridae (e.g.,
paramyxovirus, parainfluenza virus 1, mobillivirus (e.g., measles
virus), rubulavirus (e.g., mumps virus), pneumonovirinae (e.g.,
pneumovirus, human respiratory synctial virus), and metapneumovirus
(e.g., avian pneumovirus and human metapneumovirus)),
picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus (e.g.,
human hepatits A virus), cardiovirus, and apthovirus), reoviridae
(e.g., orthoreovirus, orbivirus, rotavirus, cypovirus, fijivirus,
phytoreovirus, and oryzavirus), retroviridae (e.g., mammalian type
B retroviruses, mammalian type C retroviruses, avian type C
retroviruses, type D retrovirus group, BLV-HTLV retroviruses,
lentivirus (e.g. human immunodeficiency virus 1 and human
immunodeficiency virus 2), spumavirus), flaviviridae (e.g.,
hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus),
togaviridae (e.g., alphavirus (e.g., sindbis virus) and rubivirus
(e.g., rubella virus)), rhabdoviridae (e.g., vesiculovirus,
lyssavirus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus),
arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus,
Ippy virus, and lassa virus), and coronaviridae (e.g., coronavirus
and torovirus). Examples of bacterial pathogens include but are not
limited to: but not limited to, the Aquaspirillum family,
Azospirillum family, Azotobacteraceae family, Bacteroidaceae
family, Bartonella species, Bdellovibrio family, Campylobacter
species, Chlamydia species (e.g., Chlamydia pneumoniae),
clostridium, Enterobacteriaceae family (e.g., Citrobacter species,
Edwardsiella, Enterobacter aerogenes, Erwinia species, Escherichia
coli, Hafnia species, Klebsiella species, Morganella species,
Proteus vulgaris, Providencia, Salmonella species, Serratia
marcescens, and Shigella flexneri), Gardinella family, Haemophilus
influenzae, Halobacteriaceae family, Helicobacter family,
Legionallaceae family, Listeria species, Methylococcaceae family,
mycobacteria (e.g., Mycobacterium tuberculosis), Neisseriaceae
family, Oceanospirillum family, Pasteurellaceae family,
Pneumococcus species, Pseudomonas species, Rhizobiaceae family,
Spirillum family, Spirosomaceae family, Staphylococcuss (e.g.,
methicillin resistant Staphylococcus aureus and Staphylococcus
pyrogenes), Streptococcus (e.g., Streptococcus enteritidis,
Streptococcus fasciae, and Streptococcus pneumoniae), Vampirovibrio
Helicobacter family, and Vampirovibrio family. Examples of fungal
pathogens include, but are not limited to: Absidia species (e.g.,
Absidia corymbifera and Absidia ramosa), Aspergillus species,
(e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus
nidulans, Aspergillus niger, and Aspergillus terreus), Basidiobolus
ranarum, Blastomyces dermatitidis, Candida species (e.g., Candida
albicans, Candida glabrata, Candida kerr, Candida krusei, Candida
parapsilosis, Candida pseudotropicalis, Candida quillermondii,
Candida rugosa, Candida stellatoidea, and Candida tropicalis),
Coccidioides immitis, Conidiobolus species, Cryptococcus neoforms,
Cunninghamella species, dermatophytes, Histoplasma capsulatum,
Microsporum gypseum, Mucor pusillus, Paracoccidioides brasiliensis,
Pseudallescheria boydii, Rhinosporidium seeberi, Pneumocystis
carinii, Rhizopus species (e.g., Rhizopus arrhizus, Rhizopus
oryzae, and Rhizopus microsporus), Saccharomyces species,
Sporothrix schenckii, zygomycetes, and classes such as Zygomycetes,
Ascomycetes, the Basidiomycetes, Deuteromycetes, and Oomycetes.
[0357] In another embodiment, the invention provides methods for
preventing, managing, treating or ameliorating cancer, autoimmune,
inflammatory or infectious diseases or one or more symptoms
thereof, said methods comprising administering to a subject in need
thereof a dose of a prophylactically or therapeutically effective
amount of one or more scaffolds of the invention in combination
with surgery, alone or in further combination with the
administration of a standard or experimental chemotherapy, a
hormonal therapy, a biological therapy/immunotherapy and/or a
radiation therapy. In accordance with these embodiments, the
scaffolds of the invention utilized to prevent, manage, treat or
ameliorate cancer, autoimmune, inflammatory or infectious diseases
or one or more symptoms or one or more symptoms thereof may or may
not be conjugated or fused to a moiety (e.g., therapeutic agent or
drug).
[0358] The invention provides methods for preventing, managing,
treating or ameliorating cancer, autoimmune, inflammatory or
infectious diseases or one or more symptoms or one or more symptoms
thereof, said methods comprising administering to a subject in need
thereof one or more scaffolds of the invention in combination with
one or more of therapeutic agents that are not cancer therapeutics
(a.k.a., non-cancer therapies). Examples of such agents include,
but are not limited to, anti-emetic agents, anti-fungal agents,
anti-bacterial agents, such as antibiotics, anti-inflammatory
agents, and anti-viral agents. Non-limiting examples of anti-emetic
agents include metopimazin and metochlopramide. Non-limiting
examples of antifungal agents include azole drugs, imidazole,
triazoles, polyene, amphotericin and ryrimidine. Non-limiting
examples of anti-bacterial agents include dactinomycin, bleomycin,
erythromycin, penicillin, mithramycin, cephalosporin, imipenem,
axtreonam, vancomycin, cycloserine, bacitracin, chloramphenicol,
clindamycin, tetracycline, streptomycin, tobramycin, gentamicin,
amikacin, kanamycin, neomycin, spectinomycin, trimethoprim,
norfloxacin, refampin, polymyxin, amphotericin B, nystatin,
ketocanazole, isoniazid, metronidazole and pentamidine.
Non-limiting examples of antiviral agents include nucleoside
analogs (e.g., zidovudine, acyclivir, gangcyclivir, vidarbine,
idoxuridine, trifluridine and ribavirin), foscaret, amantadine,
rimantadine, saquinavir, indinavir, ritonavir, interferon
("IFN")-.alpha.,.beta. or .gamma. and AZT. Non-limiting examples of
anti-inflammatory agents include non-steroidal anti-inflammatory
drugs ("NSAIDs"), steroidal anti-inflammatory drugs, beta-agonists,
anti-cholingenic agents and methylxanthines.
[0359] In another embodiment, the invention comprises compositions
capable of inhibiting a cancer cell phenotype. In one embodiment,
the cancer cell phenotype is cell growth, cell attachment, loss of
cell attachment, decreased receptor expression (such as, for
example, but not limited to Eph receptors), increased receptor
expression (such as, for example, but not limited to Eph
receptors), metastatic potential, cell cycle inhibition, receptor
tyrosine kinase activation/inhibition or others.
[0360] In one embodiment, the invention comprises compositions
capable of treating chronic inflammation. In one embodiment, the
compositions are useful in the targeting of immune cells for
destruction or deactivation. In one embodiment, the compositions
are useful in targeting activated T cells, dormant T cells, B
cells, neutrophils, eosiniphils, basophils, mast cells, or
dendritic cells. In another embodiment, the invention comprises
compositions capable of decreasing immune cell function. In another
embodiment, the compositions are capable of ablating immune cell
function.
[0361] In another embodiment, the invention comprises compositions
capable of inhibiting or reducing angiogenesis. In another
embodiment, the angiogenesis is related to tumor growth, rheumatoid
arthritis, SLE, Sjogren's syndrome or others.
[0362] In another embodiment, the invention comprises compositions
useful for treatment of diseases of the gastrointestinal tract. The
scaffolds of the invention exhibit a high level of stability under
low pH conditions. The stability at low pH suggests that the
composition will be suitable for oral administration for a variety
of gastrointestinal disorders, such as irritable bowel syndrome,
gastroesophageal reflux, intestinal pseudo-obstructions, dumping
syndrome, intractable nausea, peptic ulcer, appendicitis, ischemic
colitis, ulcerative colitis, gastritis, Helico pylori disease,
Crohn's disease, Whipple's disease, celiac sprue, diverticulitis,
diverticulosis, dysphagia, hiatus hernia, infections esophageal
disorders, hiccups, rumination and others.
[0363] The invention further provides combinatorial compositions
and methods of using such compositions in the prevention,
treatment, reduction, or amelioration of disease or symptoms
thereof. The scaffolds of the invention may be combined with
conventional therapies suitable for the prevention, treatment,
reduction or amelioration of disease or symptoms thereof. Exemplary
conventional therapies can be found in the Physician's Desk
Reference (56th ed., 2002 and 57th ed., 2003). In some embodiments,
scaffolds of the invention may be combined with chemotherapy,
radiation therapy, surgery, immunotherapy with a biologic (antibody
or peptide), small molecules, or another therapy known in the art.
In some embodiments, the combinatorial therapy is administered
together. In other embodiments, the combinatorial therapy is
administered separately.
[0364] The invention also provides methods of diagnosing diseases.
The scaffolds of the invention which bind a specific target
associated with a disease may be implemented in a method used to
diagnose said disease. In one embodiment, the scaffolds of the
invention are used in a method to diagnose a disease in a subject,
said method comprising obtaining a sample from the subject,
contacting the target with the scaffold in said sample under
conditions that allow the target:scaffold interaction to form,
identifying the target:scaffold complex and thereby detecting the
target in the sample. In some embodiments, the target is an antigen
associated with disease. In another embodiment, the target is a
cytokine, inflammatory mediator, and intracellular antigen, a self
antigen, a non-self antigen, an intranuclear antigen, a
cell-surface antigen, a bacterial antigen, a viral antigen or a
fungal antigen. In other embodiments, the disease to be diagnosed
is described herein.
[0365] The invention also provides methods of imaging specific
targets. In one embodiment, scaffolds of the invention conjugated
to imaging agents such as green-fluorescent proteins, other
fluorescent tags (Cy3, Cy5, Rhodamine and others), biotin, or
radionuclides may be used in methods to image the presence,
location, or progression of a specific target. In some embodiments,
the method of imaging a target comprising a scaffold of the
invention is performed in vitro. In other embodiments, the method
of imaging a target comprising a scaffold of the invention is
performed in vivo. In other embodiments, the method of imaging a
target comprising a scaffold of the invention is performed by MM,
PET scanning, X-ray, fluorescence detection or by other detection
methods known in the art.
[0366] The invention also provides methods of monitoring disease
progression, relapse, treatment, or amelioration using the
scaffolds of the invention. In one embodiment, methods of
monitoring disease progression, relapse, treatment, or amelioration
is accomplished by the methods of imaging, diagnosing, or
contacting a compound/target with a scaffold of the invention as
presented herein.
6.19 Kits
[0367] Also within the scope of the invention are kits comprising
the compositions (e.g. scaffolds) of the invention and instructions
for use. The kit can further contain a least one additional
reagent, or one or more additional scaffolds of the invention. Kits
typically include a label indicating the intended use of the
contents of the kit. The term label includes any writing, or
recorded material supplied on or with the kit, or which otherwise
accompanies the kit.
6.20 Equivalents
[0368] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0369] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference. Additionally,
U.S. Provisional Application No. 60/984,209 filed Oct. 31, 2007 is
hereby incorporated by reference herein in its entirety for all
purposes.
6.21 Exemplary Embodiments:
[0370] 1. A recombinant, non-naturally occurring polypeptide
scaffold comprising, [0371] I. a plurality of beta strand domains
linked to a plurality of loop region sequences derived from a
naturally occurring protein sequence, [0372] II. wherein one or
more of said loop region sequences vary by deletion, substitution
or addition by at least one amino acid from the corresponding loop
sequences in the naturally occurring protein sequence, and; [0373]
III. wherein the beta strand domains of the polypeptide scaffold
have at least 50%, at least 60%, at least 70%, at least 80%, or at
least 90% homology to the corresponding domain sequences of the
naturally occurring protein sequence of SEQ ID NO. 1 2. The
scaffold of embodiment 1, wherein said plurality of beta strands is
at least seven strands. 3. The scaffold of embodiment 1, wherein
said plurality of loop regions is at least six regions. 4. The
scaffold of embodiment 1, wherein said scaffold comprises seven
beta strands wherein each is designated A, B, C, D, E, and F, and
six loop regions, wherein a loop region connects each beta strand
and is designated AB, BC, CD, DE, EF, and FG loops. 5. The scaffold
of embodiment 4, wherein said scaffold comprises an AB loop
sequence of SEQ ID NO:201, a BC loop sequence of SEQ ID NO:202, a
CD loop sequence of SEQ ID NO:203, a DE loop sequence of SEQ ID
NO:204, an EF loop sequence of SEQ ID NO:205 and an FG loop
sequence of SEQ ID NO:206. 6. The scaffold of embodiment 4, wherein
said scaffold comprises an AB loop sequence of SEQ ID NO:207, a BC
loop sequence of SEQ ID NO:202, a CD loop sequence of SEQ ID
NO:203, a DE loop sequence of SEQ ID NO:208, an EF loop sequence of
SEQ ID NO:209 and an FG loop sequence of SEQ ID NO:206. 7. The
scaffold of embodiment 1, wherein said beta strand domain comprises
the polypeptide sequence encoded by SEQ ID NO.1 8. The scaffold of
embodiment 5 or 6, wherein said loop region sequences comprise the
BC and FG loops. 9. The scaffold of embodiment 8, wherein said BC
loop comprises 9 amino acids having a consensus sequence of
S-X-a-X-b-X-X-X-G, wherein X represents any amino acid, wherein (a)
represents proline or alanine and wherein (b) represents alanine or
glycine. 10. The scaffold of embodiment 8, wherein said BC loop
comprises 11 amino acids having a consensus sequence of
S-P-c-X-X-X-X-X-X-T-G, wherein X represents any amino acid and
wherein (c) represents proline, serine or glycine. 11. The scaffold
of embodiment 8, wherein said BC loop comprises 12 amino acids
having a consensus sequence of A-d-P-X-X-X-e-f-X-I-X-G, wherein X
represents any amino acid, wherein (d) represents proline,
glutamate or lysine, wherein (e) represents asparagine or glycine,
and wherein (f) represents serine or glycine. 12. The scaffold of
embodiment 8, wherein said FG loop comprises 9 amino acids having a
consensus sequence of X-a-X-X-G-X-X-X-S, wherein X represents any
amino acid and wherein (a) represents asparagine, threonine or
lysine. 13. The scaffold of embodiment 8, wherein said FG loop
comprises 10 amino acids having a consensus sequence of
X-a-X-X-X-X-b-N-P-A, wherein X represents any amino acid, wherein
(a) represents asparagine, threonine or lysine and wherein (b)
represents serine or glycine. 14. The scaffold of embodiment 8,
wherein said FG loop comprises 11 amino acids having a consensus
sequence of X-a-X-X-G-X-X-S-N-P-A, wherein X represents any amino
acid, and wherein (a) represents asparagine, threonine or lysine.
15. The scaffold of any of embodiments 4-14, wherein said loop
region sequences further comprise the DE loop. 16. The scaffold of
embodiment 15, wherein said DE loop comprises 6 amino acids having
a consensus sequence of X-X-X-X-X-X, wherein X represents any amino
acid. 17. The scaffold of any of embodiments 4-14, wherein said
loop region sequences further comprise the AB loop. 18. The
scaffold of embodiment 7 wherein said AB loop comprises 7 residues
having the consensus sequence: K-X-X-X-X-X-a, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine, and
wherein (a) represents serine, threonine, alanine, or glycine. 19.
The scaffold of embodiment 18 wherein said AB loop comprises 9
residues having the consensus sequence: K-X-X-X-X-X-X-X-a, wherein
X represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, and wherein (a) represents serine, threonine,
alanine, or glycine. 20. The scaffold of any of embodiments 4-14,
wherein said loop region sequences further comprise the CD loop.
21. The scaffold of embodiment 20, wherein said CD loop comprises
7, 8, or 9 residues wherein each residue in the CD loop is
randomized and wherein each residue may be asparagine, aspartic
acid, histidine, tyrosine, isoleucine, valine, leucine,
phenylalanine, threonine, alanine, proline, or serine. 22. The
scaffold of any of embodiments 4-14, wherein said loop region
sequences further comprise the EF loop. 23. The scaffold of
embodiment 22, wherein said EF loop comprises 8 having the
consensus sequence X-b-L-X-P-X-c-X, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine,
wherein (b) represents asparagine, lysine, arginine, aspartic acid,
glutamic acid, or glycine, and wherein (c) represents isoleucine,
threonine, serine, valine, alanine, or glycine. 24. The scaffold of
any of embodiments 1 to 14, wherein said scaffold further comprises
at least one disulfide bond. 25. The scaffold of embodiment 24,
wherein said disulfide bond forms a link between the A strand and B
strand. 26. The scaffold of embodiment 24, wherein said disulfide
bond forms a link between the D strand and E strand. 27. The
scaffold of embodiment 24, wherein said disulfide bond forms a link
between the F strand and G strand. 28. The scaffold of embodiment
24, wherein said disulfide bond forms a link between the C strand
and F strand. 29. The scaffold of any of embodiments 1 to 28,
wherein said scaffold further comprises at least two disulfide
bonds. 30. The scaffold of embodiment 29, wherein said disulfide
bonds form a first link between the F strand and the G strand, and
a second link between the C strand and F strand. 31. The scaffold
of embodiment 24 or 29, wherein said at least one disulfide bond is
in a beta strand domain. 32. The scaffold of embodiment 24 or 29,
wherein said at least one disulfide bond is in a loop region. 33.
The scaffold of embodiment 15, wherein said scaffold comprises the
sequence corresponding to Tn3.sup.SS1(SEQ ID NO:64),
Tn3.sup.SS2(SEQ ID NO:210), Tn3.sup.SS3(SEQ ID NO:65), or
Tn3.sup.SS4(SEQ ID NO:66), 34. The scaffold of embodiment 17,
wherein said scaffold comprises the sequence corresponding to
Tn3.sup.SS3+4 (SEQ ID NO:67), 35. The scaffold of any of
embodiments 1-34, wherein said scaffold binds a target. 36. The
scaffold of embodiment 35 wherein said scaffold binds said target
with an affinity (K.sub.D) of at least 100 .mu.M. 37. The scaffold
of embodiment 36, wherein said target is a cell-surface antigen, a
soluble antigen, an immobilized antigen, an immunosilent antigen,
an intracellular antigen, an intranuclear antigen, a self antigen,
a non-self antigen, a cancer antigen, a bacterial antigen, or a
viral antigen. 38. The scaffold of embodiment 36, wherein said
scaffold exhibits a thermal melting temperature (Tm) of at least
40.degree. C. as measured by Differential scanning calorimetry
(DSC) in 20 mM sodium phosphate, pH 7.0. 39. The scaffold of
embodiment 36, wherein said scaffold exhibits an increased Cm of at
least 10% as measured in a urea denaturation experiment compared to
the same scaffold prior to engineering, under similar experimental
conditions. 40. The scaffold of embodiment 36, wherein said
scaffold exhibits an increased Cm of at least 10% as measured in a
guanidine denaturation experiment compared to the same scaffold
prior to engineering, under similar experimental conditions. 41.
The scaffold of embodiment 36, wherein said scaffold exhibits an
increased resistance to protease degradation by at least 10% as
compared to the same scaffold prior to engineering under similar
experimental conditions. 42. The scaffold of embodiment 36, wherein
said scaffold is conjugated to a heterologous agent, wherein said
agent is selected from the group consisting of another scaffold,
Polyethylene glycol (PEG), human serum albumin (HSA), an Fc region
of an antibody, an IgG molecule, a binding peptide, cytotoxic drug,
radiolabel, imaging agent, His-Tag, Biotin, Flag-tag, nucleic acid,
or a cytokine. 43. A multimeric scaffold comprising at least two
scaffolds of embodiment 36. 44. The multimeric scaffold of
embodiment 43, wherein said multimeric scaffolds further comprises
an epitope binding domain, wherein said epitope binding domain is
selected from the group consisting of an antibody, antibody
fragment, diabody, scFv, Fab, Fv, a binding peptide. 45. The
multimeric scaffold of embodiment 43, wherein said epitope binding
domain is specific for a different target than said scaffold. 46.
The multimeric scaffold of embodiment 43, wherein said epitope
binding domain is specific for the same target as that of said
scaffold. 47. The multimeric scaffold of any of embodiments 43-46,
wherein said scaffolds are linked by another scaffold, an IgG
molecule or fragment thereof, an Fc region, a dimerization domain,
a chemical crosslink, a disulfide bond, or an amino acid linker.
48. An isolated nucleic acid molecule encoding the multimeric
scaffold of any of embodiments 1-47. 49. An expression vector
operably linked to the nucleic acid of embodiment 48. 50. A host
cell comprising the vector of embodiment 49. 51. A polypeptide
display library comprising the scaffold of embodiment 36, each
scaffold comprising a plurality of beta strand domains linked to a
plurality of loop region sequences derived from a naturally
occurring protein sequence, wherein one or more of said loop region
sequences vary by deletion, substitution or addition by at least
one amino acid from the corresponding loop sequences in the
naturally occurring protein sequence and wherein the beta strand
domains of the polypeptide scaffold have at least 50% homology to
the corresponding domain sequences of the naturally occurring
protein sequence of SEQ ID No:1. 52. The library of embodiment 51,
wherein said scaffold comprises at least two loop region sequences
that vary by deletion, substitution or addition by at least one
amino acid from the corresponding loop sequences in the naturally
occurring protein sequence. 53. The library of embodiment 52,
wherein said two loop region sequences are selected from the group
consisting of BC/DE, BC/FG, DE/FG, AB/CD, AB/EF, and CD/ED loops.
54. The library of embodiment 53, wherein said loop region
sequences comprise the BC and FG loops. 55. The library of
embodiment 53 wherein said BC loop comprises 9 amino acids having a
consensus sequence of S-X-a-X-b-X-X-X-G, wherein X represents any
amino acid, wherein (a) represents proline or alanine and wherein
(b) represents alanine or glycine. 56. The library of embodiment
53, wherein said BC loop comprises 11 amino acids having a
consensus sequence of S-P-c-X-X-X-X-X-X-T-G, wherein X represents
any amino acid and wherein (c) represents proline, serine or
glycine. 57. The library of embodiment 53, wherein said BC loop
comprises 12 amino acids having a consensus sequence of
A-d-P-X-X-X-e-f-X-I-X-G, wherein X represents any amino acid,
wherein (d) represents proline, glutamate or lysine, wherein (e)
represents asparagine or glycine, and wherein (f) represents serine
or glycine. 58. The library of embodiment 53, wherein said FG loop
comprises 9 amino acids having a consensus sequence of
X-a-X-X-G-X-X-X-S, wherein X represents any amino acid and wherein
(a) represents asparagine, threonine or lysine. 59. The library of
embodiment 53, wherein said FG loop comprises 10 amino acids having
a consensus sequence of X-a-X-X-X-X-b-N-P-A, wherein X represents
any amino acid, wherein (a) represents asparagine, threonine or
lysine and wherein (b) represents serine or glycine. 60. The
library of embodiment 53, wherein said FG loop comprises 11 amino
acids having a consensus sequence of X-a-X-X-G-X-X-S-N-P-A, wherein
X represents any amino acid, and wherein (a) represents asparagine,
threonine or lysine. 61. The library of embodiment 52, wherein said
loop region sequences further comprise the DE loop. 62. The library
of embodiment 61, wherein said DE loop comprises 6 amino acids
having a consensus sequence of X-X-X-X-X-X, wherein X represents
any amino acid. 63. The library of embodiment 52, wherein said loop
region sequences further comprise the AB loop. 64. The library of
embodiment 63, wherein said AB loop comprises 7 residues having the
consensus sequence: K-X-X-X-X-X-a, wherein X represents asparagine,
aspartic acid, histidine, tyrosine, isoleucine, valine, leucine,
phenylalanine, threonine, alanine, proline, or serine, and wherein
(a) represents serine, threonine, alanine, or glycine. 65. The
library of embodiment 63, wherein said AB loop comprises 9 residues
having the consensus sequence: K-X-X-X-X-X-X-X-a, wherein X
represents asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine, and wherein (a) represents serine, threonine,
alanine, or glycine. 66. The library of embodiment 52, wherein said
loop region sequences further comprise the CD loop. 67. The library
of embodiment 66 wherein said CD loop comprises 7, 8, or 9 residues
wherein each residue in the CD loop is randomized and wherein each
residue may be asparagine, aspartic acid, histidine, tyrosine,
isoleucine, valine, leucine, phenylalanine, threonine, alanine,
proline, or serine. 68. The library of embodiment 52, wherein said
loop region sequences further comprise the EF loop. 69. The library
of embodiment 68, wherein said EF loop comprises 8 having the
consensus sequence X-b-L-X-P-X-c-X, wherein X represents
asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine,
leucine, phenylalanine, threonine, alanine, proline, or serine,
wherein (b) represents asparagine, lysine, arginine, aspartic acid,
glutamic acid, or glycine, and wherein (c) represents isoleucine,
threonine, serine, valine, alanine, or glycine. 70. The library of
embodiment 51, wherein said scaffold comprises at least three loop
region sequences that vary by deletion, substitution or addition by
at least one amino acid from the corresponding loop sequences in
the naturally occurring protein sequence. 71. The library of
embodiment 70, wherein said loop region sequences comprise the BC,
DE, and FG loops. 72. The library of embodiment 70, wherein said
loop region sequences comprise the AB, CD, and EF loops. 73. The
library of embodiment 70, wherein said loop region sequences
comprise any three loops selected from the group consisting of the
AB, BC, CD, DE, EF, and FG loops.
74. The polypeptide display library of embodiment 38, wherein said
polypeptide is displayed on the surface of a ribosome,
bacteriophage, virus, bacteria, or yeast. 75. A collection of
isolated nucleic acid molecules encoding the library of any of
embodiments 51-74. 76. An expression vector operably linked to the
nucleic acid molecules of embodiment 75. 77. A method of obtaining
a polypeptide scaffold that binds to a target, said method
comprising (a) contacting a target ligand with the library of
embodiment 38 under conditions that allow a scaffold:target ligand
complex to form, and (b) obtaining from the complex, the scaffold
that binds the target ligand. 78. The method of embodiment 77,
further comprising randomizing at least one loop of said scaffold
of the protein obtained in step (b) to generate a further
randomized scaffold and repeating steps (a) and (b) using said
further randomized scaffold. 79. The method of embodiment 78,
wherein said method comprises at least two loops. 80. The method of
embodiment 78, wherein said method comprises at least three loops.
81. The method of embodiment 78, further comprising randomizing at
least one loop of said scaffold of the protein obtained in step (b)
to generate a further randomized scaffold and repeating steps (a)
and (b) using said further randomized scaffold, wherein said
repetition of steps (a) and (b) further comprises contacting a
target distinct from the target of said first operation of step (a)
and (b). 82. The method of embodiment 78, further comprising
randomizing at least one loop of said scaffold of the protein
obtained step (b), wherein said loop was not randomized in said
library to generate a further randomized scaffold and repeating
steps (a) and (b) using said further randomized scaffold. 83. The
method of embodiment 78, further comprising randomizing at least
one loop of said scaffold of the protein obtained step (b), wherein
said loop was not randomized in said library to generate a further
randomized scaffold and repeating steps (a) and (b) using said
further randomized scaffold, wherein said repetition of steps (a)
and (b) further comprises contacting a target distinct from the
target of said first operation of step (a) and (b). 84. The method
of any of embodiments 77-83 wherein said method comprises a first
randomized loop selected from the group consisting of BC, DE, and
FG loops and a second loop not randomized in said library selected
from the group consisting of AB, CD, and EF loops. 85. The method
of any of embodiments 77-83 wherein said method comprises a first
randomized loop selected from the group consisting of AB, CD, EF
loops and a second loop not randomized in said library selected
from the group consisting of BC, DE, and FG loops. 86. The method
of any of embodiments 77-83, wherein said method further comprising
randomizing at least one beta strand of said scaffold of the
protein obtained in step (b) to generate a further randomized
scaffold and repeating steps (a) and (b) using said further
randomized scaffold. 87. The method of embodiment 86, said method
further comprising at least two, three, four, five, six or seven
beta strands of said scaffold of the protein obtained in step (b)
to generate a further randomized scaffold and repeating steps (a)
and (b) using said further randomized scaffold. 88. A method of
obtaining at least two scaffolds that bind to a target said method
comprising (a) contacting a target ligand with the library of
embodiment 38 under conditions that allow a scaffold:target ligand
complex to form, (b) engaging said scaffolds with a crosslinking
agent wherein the crosslinking of said scaffolds elicits a
detectable response and (c) obtaining from the complex, said
scaffolds that bind the target. 89. The method of embodiment 88,
wherein said scaffolds recognize the same epitope. 90. The method
of embodiment 88, wherein said scaffolds recognize distinct
epitopes. 91. The method of embodiment 88, wherein said
crosslinking agent is selected from the group consisting of an
antibody, an antibody fragment, a dimerization motif, a chemical
crosslinker, a binding peptide, or an epitope tag. 92. A method of
detecting a compound in a sample, said method comprising contacting
said sample with a scaffold of any of embodiments 1-47 under
conditions that allow the formation of a compound:scaffold complex
and detecting said complex, thereby detecting said compound in said
sample. 93. A method of capturing a compound in a sample, said
method comprising contacting said sample with an immobilized
scaffold of any of embodiments 1-47 under conditions that allow the
formation of a compound:scaffold complex and removing said
immobilized scaffold, thereby capturing said compound in said
sample. 94. A sterile, pyrogen-free composition comprising the
polypeptide of any of embodiments 1 to 47. 95. A pharmaceutical
composition comprising the polypeptide of any of embodiments 1 to
47. 96. A method of preventing, treating, managing, or ameliorating
a disease in a patient with the composition of embodiment 95. 97. A
method of diagnosing or imaging a disease in a patient with the
composition of embodiment 95 or 96. 98. The method of embodiment
96, wherein said method further comprises an additional therapy,
wherein said therapy is immunotherapy, biological therapy,
chemotherapy, radiation therapy, or small molecule drug therapy.
99. The method of any of embodiments 96-98 wherein said disease is
an autoimmune disease, inflammatory disease, proliferative disease,
infectious disease, respiratory disease, gastrointestinal disease,
diabetes, lupus, or obesity. 100. A recombinant, non-naturally
occurring polypeptide scaffold comprising, [0374] I. a plurality of
predicted beta strand domains linked to a plurality of predicted
loop region sequences derived from a naturally occurring protein
sequence, [0375] II. wherein one or more of said loop region
sequences vary by deletion, substitution or addition by at least
one amino acid from the corresponding loop sequences in the
naturally occurring protein sequence, and; [0376] III. wherein the
beta strand domains of the polypeptide scaffold have at least 50%,
at least 60%, at least 70%, at least 80%, or at least 90% homology
to the corresponding domain sequences of the naturally occurring
protein sequence of SEQ ID NO.2. 101. The scaffold of embodiment
100, wherein said beta strand domains comprises the polypeptide
sequence encoded by SEQ ID NO. 2. 102. A recombinant, non-naturally
occurring polypeptide scaffold comprising, [0377] I. a plurality of
predicted beta strand domains linked to a plurality of predicted
loop region sequences derived from a naturally occurring protein
sequence, [0378] II. wherein one or more of said loop region
sequences vary by deletion, substitution or addition by at least
one amino acid from the corresponding loop sequences in the
naturally occurring protein sequence, and; [0379] III. wherein the
beta strand domains of the polypeptide scaffold have at least 50%,
at least 60%, at least 70%, at least 80%, or at least 90% homology
to the corresponding domain sequences of the naturally occurring
protein sequence of i ID No:3 or 4. 103. The scaffold of embodiment
102, wherein said beta strand domains comprises the protein
sequence of Seq ID No:3 or 4. 104. A recombinant, non-naturally
occurring polypeptide scaffold comprising, [0380] I. a plurality of
predicted beta strand domains linked to a plurality of predicted
loop region sequences derived from a naturally occurring protein
sequence, [0381] II. wherein one or more of said loop region
sequences vary by deletion, substitution or addition by at least
one amino acid from the corresponding loop sequences in the
naturally occurring protein sequence, and; [0382] III. wherein the
beta strand domains of the polypeptide scaffold have at least 50%
homology to the corresponding domain sequences of the naturally
occurring protein sequence, wherein said naturally occurring
protein sequence is selected from SEQ ID NOs: 5-32, 68-88. 105. The
scaffold of any of embodiments 100-104, wherein said plurality of
beta strands is at least seven strands. 106. The scaffold of any of
embodiments 100-104, wherein said plurality of loop regions is at
least six regions. 107. The scaffold of embodiment 100-104, wherein
said scaffold comprises seven beta strands, wherein each is
designated A, B, C, D, E, and F, and six loop regions, wherein a
loop region connects each beta strand and is designated AB, BC, CD,
DE, EF, and FG loops. 108. The scaffold of any of embodiments
100-107, wherein said scaffold further comprises at least one
disulfide bond. 109. The scaffold of any of embodiments 100-107,
wherein said scaffold further comprises at least two, three, four,
or more disulfide bonds. 110. The scaffold of embodiment 108 or
109, wherein said at least one disulfide bond is in a beta strand
domain. 111. The scaffold of embodiment 108 or 109, wherein said at
least one disulfide bond is in a loop region. 112. The scaffold of
any of embodiments 100-111, wherein said scaffold binds a target.
113. The scaffold of embodiment 112, wherein said target is a
cell-surface antigen, a soluble antigen, an immobilized antigen, an
immunosilent antigen, an intracellular antigen, an intranuclear
antigen, a self antigen, a non-self antigen, a cancer antigen, a
bacterial antigen, or a viral antigen. 114. The scaffold of
embodiment 112, wherein said scaffold binds with an affinity of at
least a Kd of 100 .mu.M. 115. The scaffold of embodiment 112,
wherein said scaffold exhibits a melting temperature (Tm) of at
least 40.degree. C. as measured by differential scanning
calorimetry (DSC) in 20 mM sodium phosphate, pH 7.0. 116. The
scaffold of embodiment 112, wherein said scaffold exhibits an
increased Cm of at least 10% as measured in a urea denaturation
experiment compared to the same scaffold prior to engineering,
under similar experimental conditions. 117. The scaffold of
embodiment 112, wherein said scaffold exhibits an increased Cm of
at least 10% as measured in a guanidine denaturation experiment
compared to the same scaffold prior to engineering, under similar
experimental conditions. 118. The scaffold of embodiment 112,
wherein said scaffold exhibits an increased resistance to protease
degradation by at least 10% as compared to the same scaffold prior
to engineering under similar experimental conditions. 119. The
scaffold of embodiment 112, wherein said scaffold is conjugated to
a heterologus agent, wherein said agent is selected from the group
consisting of another scaffold, Polyethylene glycol (PEG), human
serum albumin (HSA), an Fc region of an antibody, an IgG molecule,
a dimerization domain, a binding peptide, cytotoxic drug,
radiolabel, imaging agent, His-Tag, Biotin, Flag-tag, nucleic acid,
or a cytokine. 120. A multimeric scaffold comprising at least two
scaffolds of any of embodiments 100-119. 121. The multimeric
scaffold of embodiment 120, wherein said multimeric scaffold
further comprises an epitope binding domain, wherein said epitope
binding domain is selected from the group consisting of an
antibody, antibody fragment, diabody, scFv, Fab, Fv, or a binding
peptide. 122. The multimeric scaffold of embodiment 121, wherein
said epitope binding domain is specific for a different target than
said scaffold. 123. The multimeric scaffold of embodiment 121,
wherein said epitope binding domain is specific for the same target
than said scaffold. 124. The multimeric scaffold of any of
embodiments 120-123, wherein said scaffolds are linked by another
scaffold, an IgG molecule or fragment thereof, an Fc region, a
dimerization domain, a chemical crosslink, a disulfide bond, or an
amino acid linker. 125. An isolated nucleic acid molecule encoding
the polypeptide of any of embodiments 100-124. 126. An expression
vector operably linked to the nucleic acid of embodiment 125. 127.
A host cell comprising the vector of embodiment 126. 128. A
polypeptide display library comprising the scaffolds of any of
embodiments 100-119, each scaffold comprising a plurality of beta
strand domains linked to a plurality of loop region sequences
derived from a naturally occurring protein sequence, wherein one or
more of said loop region sequences vary by deletion, substitution
or addition by at least one amino acid from the corresponding loop
sequences in the naturally occurring protein sequence and wherein
the beta strand domains of the polypeptide scaffold have at least
50% homology to the corresponding domain sequences of the naturally
occurring protein sequence. 129. The library of embodiment 128,
wherein said scaffold comprises at least two loop region sequences
that vary by deletion, substitution or addition by at least one
amino acid from the corresponding loop sequences in the naturally
occurring protein sequence. 130. The library of embodiment 128,
wherein said two loop region sequences comprise loop sequences from
the group selected from BC/DE, BC/FG, DE/FG, AB/CD, AB/EF, and
CD/ED loops. 131. The library of embodiment 128, wherein said
scaffold comprises at least three loop region sequences that vary
by deletion, substitution or addition by at least one amino acid
from the corresponding loop sequences in the naturally occurring
protein sequence. 132. The library of embodiment 131, wherein said
loop region sequences comprise the BC, DE, and FG loops. 133. The
library of embodiment 131, wherein said loop region sequences
comprise the AB, CD, and EF loops. 134. The library of embodiment
128, wherein said polypeptide is displayed on the surface of a
ribosome, bacteriophage, virus, bacteria, or yeast. 135. The
library of embodiment 134, wherein said library has a sequence
diversity of at least 10.sup.6. 136. A method of obtaining a
scaffold that binds to a target, said method comprising (a)
contacting the target ligand with the library of embodiment 128
under conditions that allow a scaffold:target ligand complex to
form, and (b) obtaining, from the complex, the scaffold that binds
the ligand. 137. The method of embodiment 136, further comprising
randomizing at least one loop of said scaffold of the protein
obtained in step (b) to generate a further randomized scaffold and
repeating steps (a) and (b) using said further randomized scaffold.
138. The method of embodiment 137, wherein said method comprises
randomizing at least two loops. 139. The method of embodiment 137,
wherein said method comprises randomizing at least three loops.
140. The method of embodiment 137, further comprising randomizing
at least one loop of said scaffold of the protein obtained in step
(b) to generate a further randomized scaffold and repeating steps
(a) and (b) using said further randomized scaffold, wherein said
repetition of steps (a) and (b) further comprises contacting a
target distinct from the target of said first operation of step (a)
and (b). 141. The method of embodiment 137, further comprising
randomizing at least one loop of said scaffold of the protein
obtained step (b), wherein said loop was not randomized in said
library to generate a further randomized scaffold and repeating
steps (a) and (b) using said further randomized scaffold.
142. The method of embodiment 137, further comprising randomizing
at least one loop of said scaffold of the protein obtained step
(b), wherein said loop was not randomized in said library to
generate a further randomized scaffold and repeating steps (a) and
(b) using said further randomized scaffold, wherein said repetition
of steps (a) and (b) further comprises contacting a target distinct
from the target of said first operation of step (a) and (b). 143.
The method of any of embodiments 136-142 wherein said method
comprises a first randomized loop selected from the group
consisting of BC, DE, and FG loops and a second loop not randomized
in said library selected from the group consisting of AB, CD, and
EF loops. 144. The method of any of embodiments 140-142 wherein
said method comprises a first randomized loop selected from the
group consisting of AB, CD, EF loops and a second loop not
randomized in said library selected from the group consisting of
BC, DE, and FG loops. 145. The method of any of embodiments
128-144, wherein said method further comprising randomizing at
least one beta strand of said scaffold of the protein obtained in
step (b) to generate a further randomized scaffold and repeating
steps (a) and (b) using said further randomized scaffold. 146. The
method of embodiment 145, said method further comprising at least
two, three, four, five, six or seven beta strands of said scaffold
of the protein obtained in step (b) to generate a further
randomized scaffold and repeating steps (a) and (b) using said
further randomized scaffold. 147. A method of obtaining at least
two scaffolds that bind to a target said method comprising (a)
contacting a target ligand with the library of embodiment 38 under
conditions that allow a scaffold:target ligand complex to form, (b)
engaging said scaffolds with a crosslinking agent wherein the
crosslinking of said scaffolds elicits a detectable response and
(c) obtaining from the complex, said scaffolds that bind the
target. 148. The method of embodiment 147, wherein said scaffolds
recognize the same epitope. 149. The method of embodiment 147,
wherein said scaffolds recognize distinct epitopes. 150. The method
of embodiment 147, wherein said crosslinking agent is selected from
the group consisting of an antibody, an antibody fragment, a
dimerization motif, a chemical crosslinker, a binding peptide, or
an epitope tag. 151. A method of detecting a compound in a sample,
said method comprising contacting said sample with a scaffold of
embodiment 112 under conditions that allow the formation of a
compound:scaffold complex and detecting said complex, thereby
detecting said compound in said sample. 152. A method of capturing
a compound in a sample, said method comprising contacting said
sample with an immobilized scaffold of embodiment 112 under
conditions that allow the formation of a compound:scaffold complex
and removing said immobilized scaffold, thereby capturing said
compound in said sample. 153. A method of purifying the scaffold of
embodiment 101 or 102 said method comprising heating a composition
containing said scaffold up to 70.degree. C. for at least 15
minutes, and removing aggregated compounds by centrifugation. 154.
A method of purifying the scaffold of embodiment 101 or 102, said
method comprising adjusting a composition containing said scaffold
to pH 3.0 or pH 3.5 or pH 4.0, or pH 4.5, or pH 5.0; heating the
resultant composition at 50.degree. C., or at 55.degree. C., or at
60.degree. C., or at 65.degree. C., or at 70.degree. C., for 1-30
minutes; and subsequently removing aggregated compounds by
centrifugation. 155. A sterile, pyrogen-free composition comprising
the scaffold of any of embodiments 100-124 156. A pharmaceutical
composition comprising the scaffold of any of embodiments 100-124.
157. A method of preventing, treating, or ameliorating a disease in
a patient comprising administering an effective amount of the
composition of embodiment 156 to a patient. 158. A method of
monitoring disease progression in a patient comprising:
administering the composition of embodiment 156 to a patient in
need of said monitoring; imaging the composition in said patient;
and evaluating said patient. 159. The method of embodiment 157 or
158 wherein said disease is an autoimmune disease, inflammatory
disease, proliferative disease, infectious disease, respiratory
disease, gastrointestinal disease, diabetes, lupus, or obesity.
160. A scalable process of the invention wherein said process
results in a production efficiency of at least 1 g/L of a scaffold
from any of embodiments 1-47 or 100-124. 161. The process of
embodiment 160, wherein said process comprises scaffolds that are
purified from the culture media used in the process. 162. A method
of producing a scaffold from any of embodiments 1-47 or 100-124 in
which said scaffold is produced and secreted into the culture
media. 163. The method of embodiment 162 wherein said method
comprises using an expression vector comprises an oppA signal
peptide (SEQ ID NO:227). 164. A method of assaying or detecting
binding of a scaffold to a target using cell free material obtained
from the method of embodiment 162. 165. A method of purifying a
scaffold produced by the method of embodiment 162. 166. The
scaffold of embodiment 1, wherein said scaffold specifically binds
TRAIL-R2. 167. The scaffold of embodiment 166, wherein said
TRAIL-R2 is human. 168. The scaffold of embodiment 166, wherein
said scaffold comprises a sequence derived from a TRAIL-R2 binding
scaffold selected from 2F4, 5B10, 10D9, 6F11, 8B3, 5E5, 2Hb, 7G11,
6C7, 1E03, 2B04, 1C12, 1A03, 1C10, 1B12, 2G03, 2D3, 1C06, 2F08,
1B04, 3B11, 1D8, 2A12, 1E05, 2F02, 1H05, 2A11, or 1G11. 169. The
scaffold of embodiment 166, wherein said scaffold is a multimeric
scaffold. 170. The scaffold of embodiment 168, wherein said
multimeric scaffold comprises at least two scaffold domains. 171.
The scaffold of embodiment 170, wherein said at least two scaffold
domains bind the same epitope. 172. The scaffold of embodiment 170,
wherein said at least two scaffold domains bind the same epitope.
173. The scaffold of embodiment 170, wherein at least one scaffold
domain is linked to an Fc region derived from an IgG molecule. 174.
The scaffold of embodiment 170, wherein at least one scaffold
domain is linked to a CH1 region derived from an IgG molecule. 175.
The scaffold of embodiment 170, wherein at least one scaffold
domain is linked to a CH2 region derived from an IgG molecule. 176.
The scaffold of embodiment 170, wherein at least one scaffold
domain is linked to a hinge region derived from an IgG molecule.
177. The scaffold of embodiment 170, wherein at least one scaffold
domain is linked to a Ckappa or Clambda region derived from an IgG
molecule. 178. The scaffold of embodiment 170, wherein at least one
scaffold domain is 2D3 or 1C12. 179. The scaffold of embodiment
170, wherein said scaffold comprises 1C12 linked to a hinge region
derived from an IgG molecule. 180. The scaffold of embodiment 170,
wherein said scaffold comprises 2D3 linked to a hinge region
derived from an IgG molecule. 181. The scaffold of embodiment 170,
wherein said scaffold comprises 1C12 linked to a CH1 region derived
from an IgG molecule. 182. The scaffold of embodiment 170, wherein
said scaffold comprises 2D3 linked to a CH1 region derived from an
IgG molecule. 183. The scaffold of embodiment 170, wherein said
scaffold comprises 1C12 linked to a Ckappa region of an IgG
molecule. 184. The scaffold of embodiment 170, wherein said
scaffold comprises 2D3 linked to a Ckappa region of an IgG
molecule. 185. The scaffold of any of embodiments 166-184, wherein
said scaffold comprises a BC loop sequence selected from the group
consisting of SEQ ID NO:126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177,
180, 183, 186, 189, 192, 195, and 198. 186. The scaffold of any of
claims 166-184, wherein said scaffold comprises a DE loop sequence
selected from the group consisting of SEQ ID NO: 145, 148, 151,
154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190,
193, 196, and 199. 187. The scaffold of any of embodiments 166-184,
wherein said scaffold comprises an FG loop sequence selected from
the group consisting of 127, 129, 131, 133, 135, 137, 139, 141,
143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179,
182, 185, 188, 191, 194, 197, and 200. 188. The scaffold of any of
embodiments 166-188 wherein said scaffold agonizes the TRAIL-R2
receptor upon binding. 189. The scaffold of any of embodiments
166-189 wherein said scaffold mimics the binding of TRAIL to
TRAIL-R2 receptor upon binding. 190. The scaffold of any of
embodiments 166-190 wherein said scaffold acts to dimerize the
TRAIL-R2 receptor upon binding. 191. A method of agonizing the
TRAIL-R2 receptor comprising contacting the TRAIL-R2 receptor with
a scaffold of any of embodiments 166-190. 192. The method of
embodiment 191, wherein said scaffold mimics the binding of TRAIL
to TRAIL-R2. 193. The method of embodiment 192, wherein said
scaffold acts to dimerize the TRAIL-R2 receptor. 194. A method of
reducing or inhibiting cell viability comprising contacting
TRAIL-R2 receptor on the cell with a scaffold of any of embodiments
166-190. 195. A method of activating or promoting apoptosis in a
cell comprising contacting TRAIL-R2 receptor on the cell with a
scaffold of any of embodiments 166-190. 196. A method of
preventing, treating, ameliorating, or managing cancer in a patient
in need thereof by administering a scaffold or a composition
thereof of any of embodiments 166-190. 197. The method of
embodiment 196, wherein said cancer is selected from squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer (NSCLC),
non-Hodgkin's lymphoma, blastoma, gastrointestinal cancer, renal
cancer, ovarian cancer, liver cancer, stomach cancer, bladder
cancer, hepatoma, breast cancer, colon cancer, colorectal cancer,
pancreatic cancer, endometrial carcinoma, salivary gland carcinoma,
kidney cancer, liver cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, head and neck cancer, lung
cancer, adenocarcinoma, renal cell carcinoma, or hepatocellular
carcinoma. 198. A recombinant polypeptide scaffold comprising,
[0383] I. seven beta strand domains designated A, B, C, D, E, F,
and G; [0384] II. linked to six loop regions, wherein a loop region
connects each beta strand and is designated AB, BC, CD, DE, EF, and
FG loops; [0385] III. wherein at least one loop region is a
non-naturally occurring variant of the cognate loop regions in any
of SEQ ID NOs: 1-32 or 68-88; and [0386] IV. wherein at least one
beta strand domain have at least 50%, at least 60%, at least 70%,
at least 80%, or at least 90%, at least 95%, or at least 99%
homology to the cognate beta strand domains in any of SEQ ID NOs:
1-32 or 68-88. 199. The scaffold of embodiment 198, wherein said
beta strand domains comprises the polypeptide sequence encoded by
any of SEQ ID NOs: 1-32 or 68-88. 200. The scaffold of embodiment
199, wherein said beta strand domains comprise: [0387] a. amino
acid sequences for the A beta strand (SEQ ID NO:228), B beta strand
(SEQ ID NO:229), C beta strand (SEQ ID NO:230), D beta strand (SEQ
ID NO:231), E beta strand (SEQ ID NO:232), F beta strand (SEQ ID
NO:233) and G beta strand (SEQ ID NO:234); or [0388] b. amino acid
sequences for the A beta strand (SEQ ID NO:235), B beta strand (SEQ
ID NO:229), C beta strand (SEQ ID NO:230), D beta strand (SEQ ID
NO:236), E beta strand (SEQ ID NO:232), F beta strand (SEQ ID
NO:237) and G beta strand (SEQ ID NO:234).
7. SEQUENCES
[0389] Tn3 wild type loops (first embodiment):
TABLE-US-00001 AB (SEQ ID NO: 201) = DVTDTT BC (SEQ ID NO: 202) =
FKPLAEIDG CD (SEQ ID NO: 203) = KDVPGDR DE (SEQ ID NO: 204) =
LTEDENQ EF (SEQ ID NO: 205) = GNLKPD FG (SEQ ID NO: 206) =
RRGDMSSNPA
Tn3 wild type beta strands (first embodiment)
TABLE-US-00002 A (SEQ ID NO: 228) = RLDAPSQIEVK B (SEQ ID NO: 229)
= ALITW C (SEQ ID NO: 230) = IELTYGI D (SEQ ID NO: 231) = TTID E
(SEQ ID NO: 232) = YSI F (SEQ ID NO: 233) = TEYEVSLIS G (SEQ ID NO:
234) = KETFTT
Tn3 wild type loops (second embodiment):
TABLE-US-00003 AB (SEQ ID NO: 207) = KDVTDTT BC (SEQ ID NO: 202) =
FKPLAEIDG CD (SEQ ID NO: 203) = KDVPGDR DE (SEQ ID NO: 208) =
TEDENQ EF (SEQ ID NO: 209) = GNLKPDTE FG (SEQ ID NO: 206) =
RRGDMSSNPA
Tn3 wild type beta strands (second embodiment)
TABLE-US-00004 A (SEQ ID NO: 235) = RLDAPSQIEV B (SEQ ID NO: 229) =
ALITW C (SEQ ID NO: 230) = IELTYGI D (SEQ ID NO: 236) = TTIDL E
(SEQ ID NO: 232) = YSI F (SEQ ID NO: 237) = YEVSLIS G (SEQ ID NO:
234) = KETFTT
Protein Sequences
[0390] Upper case lettering corresponds to the Tn3 structural
motif, while lower case lettering are flanking sequence appendages
derived from the synthetic cDNA and expression vector.
TABLE-US-00005 1P: SEQ ID NO: 5
aaLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKP
GVDYTITVYAVTGRGDSPASSKPISINYRTgggtlehhhhhh SEQ ID NO: 6
LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPG
VDYTITVYAVTGRGDSPASSKPISINYRT 2P: SEQ ID NO: 7
aaPTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPG
VQYNITIYAVEENQESTPVVIQQETgggtlehhhhhh SEQ ID NO: 8
PTVDQVDDTSIVVRWSRPQAPITGYRIVYSPSVEGSSTELNLPETANSVTLSDLQPGV
QYNITIYAVEENQESTPVVIQQET 3P: SEQ ID NO: 9
aaPYNTEVTETTIVITWTPAPRIGFKLGVRPSQGGEAPREVTSDSGSIVVSGLTPGVEYV
YTIQVLRDGQERDAPIVNKVVTgggtlehhhhhh SEQ ID NO: 10
PYNTEVTETTIVITWTPAPRIGFKLGVRPSQGGEAPREVTSDSGSIVVSGLTPGVEYVY
TIQVLRDGQERDAPIVNKVVT 4P: SEQ ID NO: 11
aaPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKW
KMMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPgggtlehhhhhh SEQ ID
NO: 12 PPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWK
MMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLP 5P: SEQ ID NO: 13
aaPPSLNVTKDGDSYSLRWETMKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHS
MALPALEPSTRYWARVRVRTSRTGYNGIWSEWSEARSWDTEgggtlehhhhhh SEQ ID NO: 14
PPSLNVTKDGDSYSLRWETMKMREHIDHTFEIQYRKDTATWKDSKTETLQNAHSM
ALPALEPSTRYWARVRVRTSRTGYNGIWSEWSEARSWDTE 6P: SEQ ID NO: 15
aaPPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETRITESK1V
TILHKGFSASVRTILQNDHSLLASSWASAELHAgggtlehhhhhh SEQ ID NO: 16
PPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETRITESK1VTI
LHKGFSASVRTILQNDHSLLASSWASAELHA 7P: SEQ ID NO: 17
aaLSVTDVTTSSLRLNWEAPPGAFDSFLLRFGVPSPSTLEPHPRPLLQRELMVPGTRHS
AVLRDLRSGTLYSLTLYGLRGPHKADSIQGTARTgggtlehhhhhh SEQ ID NO: 18
LSVTDVTTSSLRLNWEAPPGAFDSFLLRFGVPSPSTLEPHPRPLLQRELMVPGTRHSA
VLRDLRSGTLYSLTLYGLRGPHKADSIQGTART 8P: SEQ ID NO: 19
aaLRALNLTEGFAVLHWKPPQNPVDTYDIQVTAPGAPPLQAETPGSAVDYPLHDLVL
HTNYTATVRGLRGPNLTSPASITFTTgggtlehhhhhh SEQ ID NO: 20
LRALNLTEGFAVLHWKPPQNPVDTYDIQVTAPGAPPLQAETPGSAVDYPLHDLVLHT
NYTATVRGLRGPNLTSPASITFTT 9P: SEQ ID NO: 21
aaLEAKEVTPRTALLTWTEPPVRPAGYLLSFHTPGGQTQEILLPGGITSHQLLGLFPSTS
YNARLQAMWGQSLLPPVSTSFTTgggtlehhhhhh SEQ ID NO: 22
LEAKEVTPRTALLTWTEPPVRPAGYLLSFHTPGGQTQEILLPGGITSHQLLGLFPSTSY
NARLQAMWGQSLLPPVSTSFTT 10P: SEQ ID NO: 23
aaIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPD
TEYEVSLISRRGDMSSNPAKETFTTgggtlehhhhhh SEQ ID NO: 24
IEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDT
EYEVSLISRRGDMSSNPAKETFTT 11P: SEQ ID NO: 25
aaPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYV
SAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVgggtlehhhhhh SEQ ID NO: 26
PKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVS
AGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSV 12P: SEQ ID NO: 27
aaPSGFPQNLHVTGLTTSTTELAWDPPVLAERNGRIISYTVVFRDINSQQELQNITTDT
RFTLTGLKPDTTYDIKVRAWTSKGSGPLSPSIQSRTMPVEgggtlehhhhhh SEQ ID NO: 28
PSGFPQNLHVTGLTTSTTELAWDPPVLAERNGRIISYTVVFRDINSQQELQNITTDTRF
TLTGLKPDTTYDIKVRAWTSKGSGPLSPSIQSRTMPVE 13P: SEQ ID NO: 29
aaPKPPIDLVVTETTATSVTLTWDSGNSEPVTYYGIQYRAAGTEGPFQEVDGVATTRY
SIGGLSPFSEYAFRVLAVNSIGRGPPSEAVRARTGEQAgggtlehhhhhh SEQ ID NO: 30
PKPPIDLVVTETTATSVTLTWDSGNSEPVTYYGIQYRAAGTEGPFQEVDGVATTRYSI
GGLSPFSEYAFRVLAVNSIGRGPPSEAVRARTGE 14P: SEQ ID NO: 31
aaLSPPRNLRISNVGSNSARLTWDPTSRQINGYRIVYNNADGTEINEVEVDPITTFPLKG
LTPLTEYTIAIFSIYDEGQSEPLTGVFTTgggtlehhhhhh SEQ ID NO: 32
LSPPRNLRISNVGSNSARLTWDPTSRQINGYRIVYNNADGTEINEVEVDPITTFPLKGL
TPLTEYTIAIFSIYDEGQSEPLTGVFTT
DNA Sequences
[0391] Upper case lettering corresponds to the Tn3 structural
motif, while lower case lettering are flanking sequence
appendages.
TABLE-US-00006 1D: SEQ ID NO: 33
gccatggccgccCTGGAAGTGGTGGCGGCGACCCCGAACCAAGCCTGCTGATTAGCTGGG
ATGCGCCGGCGGTGACCGTGCGCTATTATCGTATTACCTATGGCGAAACCGGCGG
CAATAGCCCGGTGCAGGAATTTACCGTGCCGGGCAGCAAAAGCACCGCGACCAT
TAGCGGCCTGAAACCGGGCGTGGATTATACCATTACCGTGTATGCGGTGACCGGC
CGTGGCGATAGCCCGGCGAGCAGCAAACCGATTAGCATTAACTATCGTACCggtgg cggtacc
SEQ ID NO: 34
CTGGAAGTGGTGGCGGCGACCCCGACCGACCTGCTGATTAGCTGGGATGCGCCG
GCGGTGACCGTGCGCTATTATCCGTATTACCTATGGCGAAACCGGCGGCAATAGCC
CGGTGCAGGAATTTACCGTGCCGGGCAGCAAAAGCACCGCGACCATTAGCGGCC
TGAAACCGGGCGTGGATTATACCATTACCGTGTATGCGGTGACCGGCCGTGGCG
ATAGCCCGGCGAGCAGCAAACCGATTAGCATTAACTATCGTACC 2D: SEQ ID NO: 35
gccatggccgccCCGACCGTGGATCAGGTGGATGATACCAGCATTGTGGTGCGCTGGA
GCCGTCCGCAGGCGCCGATTACCGGCTATCGTATTGTGTATAGCCCGAGCGTGGA
AGGCAGCAGCACCGAACTGAACCTGCCGGAAACCGCGAATAGCGTGACCCTGAG
CGATCTGCAGCCGGGCGTGCAGTATAACATTACCATTTATGCGGTGGAAGAAAA
CCAGGAAAGCACCCCGGTGGTGATTCAGCAGGAAACCggtggcggtacc SEQ ID NO: 36
CCGACCGTGGATCAGGTGGATGATACCAGCATTGTGGTGCGCTGGAGCCGTCCG
CAGGCGCCGATTACCGGCTATCGTATTGTGTATAGCCCGAGCGTGGAAGGCAGC
AGCACCGAACTGAACCTGCCGGAAACCGCGAATAGCGTGACCCTGAGCGATCTG
CAGCCGGGCGTGCAGTATAACATTACCATTTATGCGGTGGAAGAAAACCAGGAA
AGCACCCCGGTGGTGATTCAGCAGGAAACC 3D: SEQ ID NO: 37
gccatggccgccCCGTATAACACCGAAGTGACCGAAACCACCATTGTGATTACCTGGAC
CCCGGCGCCGCGTATTGGCTTTAAACTGGGCGTGCGTCCGAGCCAGGGCGGTGA
AGCGCCGCGCGAAGTGACCAGCGATAGCGGCAGCATTGTGGTGAGCGGCCTGAC
CCCGGGCGTGGAATATGTGTATACCATTCAGGTGCTGCGTGATGGCCAGGAACGT
GATGCGCCGATTGTGAACAAAGTGGTGACCggtggcggtacc SEQ ID NO: 38
CCGTATAACACCGAAGTGACCGAAACCACCATTGTGATTACCTGGACCCCGGCG
CCGCGTATTGGCTTTAAACTGGGCGTGCGTCCGAGCCAGGGCGGTGAAGCGCCG
CGCGAAGTGACCAGCGATAGCGGCAGCATTGTGGTGAGCGGCCTGACCCCGGGC
GTGGAATATGTGTATACCATTCAGGTGCTGCGTGATGGCCAGGAACGTGATGCGC
CGATTGTGAACAAAGTGGTGACC 4D: SEQ ID NO: 39
gccatggccgccCCGCCGATCGCTCTGAATTGGACCCTGCTGAATGTTTCGCTGACCGG
TATTCATGCCGATATTCAGGTGCGTTGGGAAGCGCCGCGTAACGCCGATATTCAG
AAAGGCTGGATGGTGCTGGAATATGAACTGCAGTATAAAGAAGTGAATGAAACC
AAATGGAAAATGATGGACCCGATTCTGACCACCAGCGTGCCGGTGTACAGCCTG
AAAGTGGATAAAGAATACGAAGTCCGTGTGCGTTCTAAACAGCGTAATAGCGGC
AATTATGGTGAATTTAGTGAAGTCCTGTATGTTACCCTGCCGggtggcggtacc SEQ ID NO:
40 CCGCCGATCGCTCTGAATTGGACCCTGCTGAATGTTTCGCTGACCGGTATTCATG
CCGATATTCAGGTGCGTTGGGAAGCGCCGCGTAACGCCGATATTCAGAAAGGCT
GGATGGTGCTGGAATATGAACTGCAGTATAAAGAAGTGAATGAAACCAAATGGA
AAATGATGGACCCGATTCTGACCACCAGCGTGCCGGTGTACAGCCTGAAAGTGG
ATAAAGAATACGAAGTCCGTGTGCGTTCTAAACAGCGTAATAGCGGCAATTATG
GTGAATTTAGTGAAGTCCTGTATGTTACCCTGCCG 5D: SEQ ID NO: 41
gccatggccgccCCGCCGAGCCTGAACGTGACCAAAGATGGCGATAGCTATAGCCTGC
GCTGGGAAACCATGAAAATGCGCTATGAACATATTGATCATACCTTTGAAATTCA
GTATCGCAAAGATACCGCGACCTGGAAAGATAGCAAAACCGAAACCCTGCAGAA
CGCGCATAGCATGGCGCTGCCGGCGCTGGAACCGAGCACCCGTTATTGGGCGCG
TGTGCGTGTGCGTACCAGCCGTACCGGCTATAATGGCATTTGGAGCGAATGGAGC
GAAGCGCGTAGCTGGGATACCGAAggtggcggtacc
CCGCCGAGCCTGAACGTGACCAAAGATGGCGATAGCTATAGCCTGCGCTGGGAA
ACCATGAAAATGCGCTATGAACATATTGATCATACCTTTGAAATTCAGTATCGCA
AAGATACCGCGACCTGGAAAGATAGCAAAACCGAAACCCTGCAGAACGCGCATA
GCATGGCGCTGCCGGCGCTGGAACCGAGCACCCGTTATTGGGCGCGTGTGCGTGT
GCGTACCAGCCGTACCGGCTATAATGGCATTTGGAGCGAATGGAGCGAAGCGCG
TAGCTGGGATACCGAA 6D: SEQ ID NO: 42
gccatggccgccCCGCCGGTGAACTTTACCATTAAAGTGACCGGCCTGGCGCAGGTGCT
GCTGCAGTGGAAACCGAACCCGGATCAGGAACAGCGTAACGTGAACCTGGAATA
TCAGGTGAAAATTAACGCGCCGAAAGAAGATGATTATGAAACCCGCATTACCGA
AAGCAAACTGGTGACCATTCTGCATAAAGGCTTTAGCGCGAGCGTGCGTACCATT
CTGCAGAACGATCATAGCCTGCTGGCGAGCAGCTGGGCGAGCGCGGAACTGCAT
GCGggtggcggtacc SEQ ID NO: 43
CCGCCGGTGAACTTTACCATTAAAGTGACCGGCCTGGCGCAGGTGCTGCTGCAGT
GGAAACCGAACCCGGATCAGGAACAGCGTAACGTGAACCTGGAATATCAGGTGA
AAATTAACGCGCCGAAAGAAGATGATTATGAAACCCGCATTACCGAAAGCAAAC
TGGTGACCATTCTGCATAAAGGCTTTAGCGCGAGCGTGCGTACCATTCTGCAGAA
CGATCATAGCCTGCTGGCGAGCAGCTGGGCGAGCGCGGAACTGCATGCG 7D: SEQ ID NO: 44
gccatggccgccCTGAGCGTGACCGATGTGACCACCAGCAGCCTGCGTCTGAACTGGG
AAGCGCCGCCGGGCGCGTTTGATAGCTTTCTGCTGCGTTTTGGCGTGCCGAGCCC
GAGCACCCTGGAACCGCATCCGCGTCCGCTGCTGCAGCGTGAACTGATGGTGCC
GGGCACCCGTCATAGCGCGGTGCTGCGTGATCTGCGTAGCGGCACCCTGTATAGC
CTGACCCTGTATGGCCTGCGTGGCCCGCATAAAGCGGATAGCATTCAGGGCACC
GCGCGTACCggtggcggtacc SEQ ID NO: 45
CTGAGCGTGACCGATGTGACCACCAGCAGCCTGCGTCTGAACTGGGAAGCGCCG
CCGGGCGCGTTTGATAGCTTTCTGCTGCGTTTTGGCGTGCCGAGCCCGAGCACCC
TGGAACCGCATCCGCGTCCGCTGCTGCAGCGTGAACTGATGGTGCCGGGCACCC
GTCATAGCGCGGTGCTGCGTGATCTGCGTAGCGGCACCCTGTATAGCCTGACCCT
GTATGGCCTGCGTGGCCCGCATAAAGCGGATAGCATTCAGGGCACCGCGCGTACC 8D: SEQ ID
NO: 46 gccatggccgccCTGCGCGCGCTGAACCTGACCGAAGGCTTTGCGGTGCTGCATTGGAA
ACCGCCGCAGAACCCGGTGGATACCTATGATATTCAGGTGACCGCGCCGGGCGC
GCCGCCGCTGCAGGCGGAAACCCCGGGCAGCGCGGTGGATTATCCGCTGCATGA
TCTGGTGCTGCATACCAACTATACCGCGACCGTGCGTGGCCTGCGCGGCCCGAAT
CTGACCAGCCCGGCGAGCATTACCTTTACCACCggtggcggtacc SEQ ID NO: 47
CTGCGCGCGCTGAACCTGACCGAAGGCTTTGCGGTGCTGCATTGGAAACCGCCGC
AGAACCCGGTGGATACCTATGATATTCAGGTGACCGCGCCGGGCGCGCCGCCGC
TGCAGGCGGAAACCCCGGGCAGCGCGGTGGATTATCCGCTGCATGATCTGGTGC
TGCATACCAACTATACCGCGACCGTGCGTGGCCTGCGCGGCCCGAATCTGACCAG
CCCGGCGAGCATTACCTTTACCACC 9D: SEQ ID NO: 48
gccatggccgccCTGGAAGCGAAAGAAGTGACCCCGCGTACCGCGCTGCTGACCTGGA
CCGAACCGCCGGTGCGCCCGGCGGGTTATCTGCTGAGCTTTCATACCCCGGGCGG
CCAGACCCAGGAAATTCTGCTGCCGGGCGGCATTACCAGCCATCAGCTGCTGGG
CCTGTTTCCGAGCACCAGCTATAACGCGCGTCTGCAGGCGATGTGGGGCCAGAG
CCTGCTGCCGCCGGTGAGCACCAGCTTTACCACCggtggcggtacc SEQ ID NO: 49
CTGGAAGCGAAAGAAGTGACCCCGCGTACCGCGCTGCTGACCTGGACCGAACCG
CCGGTGCGCCCGGCGGGTTATCTGCTGAGCTTTCATACCCCGGGCGGCCAGACCC
AGGAAATTCTGCTGCCGGGCGGCATTACCAGCCATCAGCTGCTGGGCCTGTTTCC
GAGCACCAGCTATAACGCGCGTCTGCAGGCGATGTGGGGCCAGAGCCTGCTGCC
GCCGGTGAGCACCAGCTTTACCACC 10D: SEQ ID NO: 50
gccatggccgccATTGAAGTGAAAGATGTGACCGATACCACCGCGCTGATTACCTGGTT
TAAACCGCTGGCGGAAATTGATGGCATTGAACTGACCTATGGCATTAAAGATGT
GCCGGGCGATCGCACCACCATTGATCTGACCGAAGATGAAAACCAGTATAGCAT
TGGCAACCTGAAACCGGATACCGAATATGAAGTGAGCCTGATTAGCCGTCGTGG
CGATATGAGCAGCAACCCGGCGAAAGAAACCTTTACCACCggtggcggtacc SEQ ID NO: 51
ATTGAAGTGAAAGATGTGACCGATACCACCGCGCTGATTACCTGGTTTAAACCGC
TGGCGGAAATTGATGGCATTGAACTGACCTATGGCATTAAAGATGTGCCGGGCG
ATCGCACCACCATTGATCTGACCGAAGATGAAAACCAGTATAGCATTGGCAACC
TGAAACCGGATACCGAATATGAAGTGAGCCTGATTAGCCGTCGTGGCGATATGA
GCAGCAACCCGGCGAAAGAAACCTTTACCACC 11D: SEQ ID NO: 52
gccatggccgccCCGAAATTTACCAAATGCCGTAGCCCGGAACGCGAAACCTTTAGCTG
CCATTGGACCGATGAAGTTCATCATGGCACCAAAAATCTGGGCCCGATTCAGCTG
TTTTATACCCGCCGTAATACCCAGGAATGGACCCAGGAATGGAAAGAATGCCCG
GATTATGTTAGCGCGGGCGAAAACAGCTGCTATTTTAACAGCAGCTTTACCAGCA
TTTGGATTCCGTATTGCATTAAACTGACCAGCAACGGTGGCACCGTTGATGAAAA
ATGCTTTAGCGTGggtggcggtacc SEQ ID NO: 53
CCGAAATTTACCAAATGCCGTAGCCCGGAACGCGAAACCTTTAGCTGCCATTGGA
CCGATGAAGTTCATCATGGCACCAAAAATCTGGGCCCGATTCAGCTGTTTTATAC
CCGCCGTAATACCCAGGAATGGACCCAGGAATGGAAAGAATGCCCGGATTATGT
TAGCGCGGGCGAAAACAGCTGCTATTTTAACAGCAGCTTTACCAGCATTTGGATT
CCGTATTGCATTAAACTGACCAGCAACGGTGGCACCGTTGATGAAAAATGCTTTA GCGTG 12D:
SEQ ID NO: 54
gccatggcagccCCGTCTGGTTTTCCGCAGAATCTGCATGTGACCGGCCTGACCACCAG
CACCACCGAACTGGCGTGGGATCCGCCGGTGCTGGCGGAACGCAACGGCCGTAT
TATTAGCTATACCGTGGTGTTTCGTGATATTAACAGCCAGCAGGAACTGCAGAAC
ATTACCACCGATACCCGCTTTACCCTGACCGGTCTGAAACCGGATACCACCTATG
ATATTAAAGTGCGCGCCTGGACCAGCAAAGGCAGCGGCCCGCTGAGCCCGAGCA
TTCAGAGCCGCACCATGCCGGTGGAAggtggcggtacc SEQ ID NO: 55
CCGTCTGGTTTTCCGCAGAATCTGCATGTGACCGGCCTGACCACCAGCACCACCG
AACTGGCGTGGGATCCGCCGGTGCTGGCGGAACGCAACGGCCGTATTATTAGCT
ATACCGTGGTGTTTCGTGATATTAACAGCCAGCAGGAACTGCAGAACATTACCAC
CGATACCCGCTTTACCCTGACCGGTCTGAAACCGGATACCACCTATGATATTAAA
GTGCGCGCCTGGACCAGCAAAGGCAGCGGCCCGCTGAGCCCGAGCATTCAGAGC
CGCACCATGCCGGTGGAA 13D: SEQ ID NO: 56
gccatggccgccCCGAAACCGCCGATTGATCTGGTGGTTACCGAAACCACCGCGACCA
GCGTGACCCTGACCTGGGATAGCGGCAATAGCGAACCGGTGACCTATTATGGTA
TTCAGTATCGCGCGGCGGGCACCGAAGGTCCGTTTCAGGAAGTGGATGGCGTGG
CGACCACCCGTTATAGCATTGGCGGTCTGAGCCCGTTTAGCGAATATGCGTTTCG
CGTGCTGGCGGTTAATAGCATTGGCCGCGGTCCGCCGAGCGAAGCGGTGCGTGC
GCGCACCGGCGAACAGGCGggtggcggtacc SEQ ID NO: 57
CCGAAACCGCCGATTGATCTGGTGGTTACCGAAACCACCGCGACCAGCGTGACC
CTGACCTGGGATAGCGGCAATAGCGAACCGGTGACCTATTATGGTATTCAGTATC
GCGCGGCGGGCACCGAAGGTCCGTTTCAGGAAGTGGATGGCGTGGCGACCACCC
GTTATAGCATTGGCGGTCTGAGCCCGTTTAGCGAATATGCGTTTCGCGTGCTGGC
GGTTAATAGCATTGGCCGCGGTCCGCCGAGCGAAGCGGTGCGTGCGCGCACCGG CGAACAGGCG
14D: SEQ ID NO: 58
gccatggccgccCTGAGCCCGCCGCGTAACCTGCGCATTAGCAACGTGGGTAGCAATA
GCGCGCGCCTGACCTGGGATCCGACCAGCCGCCAGATTAATGGCTATCGCATTGT
GTATAACAACGCCGATGGCACCGAAATTAACGAAGTGGAAGTGGATCCGATTAC
CACCTTTCCGCTGAAAGGCCTGACCCCGCTGACCGAATATACCATTGCGATTTTT
AGCATTTATGATGAAGGTCAGAGCGAACCGCTGACCGGTGTGTTTACCACCggtggc ggtacc
SEQ ID NO: 59
CTGAGCCCGCCGCGTAACCTGCGCATTAGCAACGTGGGTAGCAATAGCGCGCGC
CTGACCTGGGATCCGACCAGCCGCCAGATTAATGGCTATCGCATTGTGTATAACA
ACGCCGATGGCACCGAAATTAACGAAGTGGAAGTGGATCCGATTACCACCTTTC
CGCTGAAAGGCCTGACCCCGCTGACCGAATATACCATTGCGATTTTTAGCATTTA
TGATGAAGGTCAGAGCGAACCGCTGACCGGTGTGTTTACCACC Wild type Tn3 domain
SEQ ID NO: 1
RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSI
GNLKPDTEYEVSLISRRGDMSSNPAKETFTT
Tenascin Tn3 Scaffold Protein Sequence
TABLE-US-00007 [0392] SEQ ID NO: 60
AAIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTED
ENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTTGGGTLEHHHHH H SEQ ID NO: 61
IEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDEN
QYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTT
Synagis Binding Tn3 Variant SynBP01
Protein Sequence
TABLE-US-00008 [0393] SEQ ID NO: 62
AAIEVKDVTDTTALITWSPPSVLVGYTIELTYGIKDVPGDRTTIDLTEDE
NQYSIGNLKPDTEYEVSLISVTEFGRRRSKETFTTGGGTLEHHHHHH SEQ ID NO: 63
IEVKDVTDTTALITWSPPSVLVGYTIELTYGIKDVPGDRTTIDLTEDENQ
YSIGNLKPDTEYEVSLISVTEFGRRRSKETFTT
Disulfide-Stabilized Tn3s (SS1-4):
TABLE-US-00009 [0394] SEQ ID NO: 64 (SS1):
IECKDVTDTTALCTWFKPLAEIDGIELTYGIKDVPGDRTTIDL
TEDENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTT SEQ ID NO: 210 (SS2):
IEVKDVTDTTALITWFKPLAEIDGIELTY IKDVP DRTTCDL
TGEDEGNQCSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTT SEQ ID NO: 65 (SS3):
IEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDL
TEDENQYSIGNLKPDTEYCVSLISRRGDMSSNPAKECFTT SEQ ID NO: 66 (SS4):
IEVKDVTDTTALITWFKPLAEIDGCELTYGIKDVPGDRTTIDL
TEDENQYSIGNLKPDTEYEVSLICRRGDMSSNPAKETFTT SEQ ID NO: 67 (SS3 + 4):
IEVKDVTDTTALITWFKPLAEIDGCELTYGIKDVPGDRT
TIDLTEDENQYSIGNLKPDTEYCVSLICRRGDMSSNPAKECFTT
Scaffolds Identified from Archea Archaeoglobus fulgidus DSM 4304
NCBI accession: NC.sub.--000917 Protein sequence:
TABLE-US-00010 SEQ ID NO: 68
PAISNVRVSDVTNSSATIRWDVSLAANNRVLFSTNSDLSSPQWSAWDN
STDSPMITLSGLSAGTAYYFSVYSFRPDNASLYSNSSIMSFTT
Staphylothermus marinus F1 NCBI accession: NC.sub.--009033 Protein
sequence:
TABLE-US-00011 Seq ID No: 2
SEPQNLKATAGNNNITLTWDPPIDDGGCRIVEYRIYRGTNNNNLEYYASV
NGSTTTFIDKNIVYSQTYYYKVSAVNNIVEGPKSNTASATPTSS
Sulfolobus acidocaldarius DSM 639 NCBI accession: NC.sub.--007181
Protein sequences:
TABLE-US-00012 1.sup.st Tn3 structural motif, SEQ ID NO: 69
PPPKPVIRFAQAGNNSISLSWYDTNTSGYYIQWWSSIDNNKSTINVGNVS
SYLFINLTNGVTYYFRIIPYNQAGNGTSSDIISLTPGAV 2.sup.nd Tn3 structural
motif, SEQ ID NO: 70
PDSPSVKVIVGDRNATVIWSKPYNGGFPILGYYLTVKTDNSSYTINVGNV
SKYTLTNLTPEVLYEVMVVAYNKLGNSSPGIVNFVALTT 3.sup.rd Tn3 structural
motif, SEQ ID NO: 71
LTTASISVSVYKKVNGVLISWNKTENTTYNLLISDKKGKIIVNITTTNTS
YFAYIPYGIYNVTIRATNQVGTNSTSFPIVFYIPPFI 4.sup.th Tn3 structural
motif, SEQ ID NO: 72
PLVKFSIGNNSILNLKWNNVTGATFYLVYVNTTLIANVTTDSYSLNLTP
GFHVIRVVAANPIYNSSPASLGILIQQHSVTSSIT
Sulfolobus solfataricus P2 NCBI accession: NC.sub.--002754 Protein
sequences:
TABLE-US-00013 1.sup.st Tn3 structural motif, SEQ ID NO: 73
PLPPKITSYSAGNESVTLGWNPVRLSSGYEIIYWNNMGFNSSINVGNV
TSYTVTGLKDGITYYFEVLAYNSIGYSSPSSIIALTPASV 2.sup.nd Tn3 structural
motif, SEQ ID NO: 74
PNPPQLVSVKYGNDNVTLNWLPPTFSGGYLLLGYYVIVKNENSMVSS
HFVNSTSLTISNLTPNVTYNVFIYAVNKLGNSSPLVLTVVPITKA 3.sup.rd Tn3
structural motif, SEQ ID NO: 75
PITKASVFAFITKLGNGILVNWTTSFPANITLELYNPNGNLISQIAAIKG
NSSYLFRVPQGNYTLVIIASNSAGVSKYVYQVVYYL 4.sup.th Tn3 structural motif,
SEQ ID NO: 76 PPASPQVSLIGFGNNLYISWNNEANVITYLVYVNNSLVYEGPSNSIVTN
ISNGTYLVKVIGVNPAGSSSPGIAVIHYTGDYVT
Sulfolobus tokodaii str. 7 NCBI accession: NC.sub.--003106 Protein
sequences:
TABLE-US-00014 1.sup.st Tn3 structural motif, Seq ID No: 3
PPKPQIASIASGNETITVKWYDTNASGYYITYWSNFSQKVTINVGNYTSY
TIKHLKDGVTYYIQIVPYNSLGNGTPSDIISATPSSV 2.sup.nd Tn3 structural
motif, Seq ID No: 4
PNPPIIKVKIGNLNATLTWYDTFNGGYPIEGYYLYVNGKGINVGNITSYV
LTNLTAGELYTIELIAYNKIGNSSISSVSFIAASKA 3.sup.rd Tn3 structural motif,
SEQ ID NO: 77 ASKANLTVTVYKKINGFLVSWNSTSKAKYILTVSKENVVLLNVSTTNTSY
FVKVPFGVYNISLEAVNIVGITKYAFILIYYIQ 4.sup.th Tn3 structural motif,
SEQ ID NO: 78 PASPTVNWSITLNTVSLNWSKVSGAEYYLIYDNGKLITNTTNTAFTFNL
TIGQNEIEVYAANAYYKSAPYIINDVRNYIVV
Protein Sequences from Example 5
[0395] Upper case lettering corresponds to the Tn3 structural
motif, while lower case lettering are flanking sequence appendages
derived from the synthetic cDNA and expression vector.
>Archaeoelobus
TABLE-US-00015 [0396] SEQ ID NO: 79
aaPAISNVRVSDVTNSSATIRWDVSLAANNRVLFSTNSDLSSPQWSAWDN
STDSPMITLSGLSAGTAYYFSVYSFRPDNASLYSNSSIMSFTTgggtleh hhhhh SEQ ID NO:
80 PAISNVRVSDVTNSSATIRWDVSLAANNRVLFSTNSDLSSPQWSAWDN
STDSPMITLSGLSAGTAYYFSVYSFRPDNASLYSNSSIMSFTT
>Staphylothermus marinus
TABLE-US-00016 SEQ ID NO: 81
aaSEPQNLKATAGNNNITLTWDPPIDDGGCRIVEYRIYRGTNNNNLEY
YASVNGSTTTFIDKNIVYSQTYYYKVSAVNNIVEGPKSNTASATPTSS gggtlehhhhhh SEQ
ID NO: 82 SEPQNLKATAGNNNITLTWDPPIDDGGCRIVEYRIYRGTNNNNLEYYA
SVNGSTTTFIDKNIVYSQTYYYKVSAVNNIVEGPKSNTASATPTSS
>Sulfolobus solfataricus
TABLE-US-00017 SEQ ID NO: 83
aaPLPPKITSYSAGNESVTLGWNPVRLSSGYEIIYWNNMGFNSSINVGN
VTSYTVTGLKDGITYYFEVLAYNSIGYSSPSSIIALTPASVgggtlehh hhhh SEQ ID NO:
84 PLPPKITSYSAGNESVTLGWNPVRLSSGYEIIYWNNMGFNSSINVGNVT
SYTVTGLKDGITYYFEVLAYNSIGYSSPSSIIALTPASV
>Sulfolobus tokodaii.sub.--1
TABLE-US-00018 SEQ ID NO: 85
aaPPKPQIASIASGNETITVKWYDTNASGYYITYWSNFSQKVTINVGN
VTSYTIKHLKDGVTYYIQIVPYNSLGNGTPSDIISATPSSVgggtlehh hhhh SEQ ID NO:
86 PPKPQIASIASGNETITVKWYDTNASGYYITYWSNFSQKVTINVGNVT
SYTIKHLKDGVTYYIQIVPYNSLGNGTPSDIISATPSSV
>Sulfolobus tokodaii.sub.--2
TABLE-US-00019 SEQ ID NO: 87
aaPNPPIIKVKIGNLNATLTWYDTFNGGYPIEGYYLYVNGKGINVGN
ITSYVLTNLTAGELYTIELIAYNKIGNSSISSVSFIAASKAgggtlehh hhhh SEQ ID NO:
88 PNPPIIKVKIGNLNATLTWYDTFNGGYPIEGYYLYVNGKGINVGNI
TSYVLTNLTAGELYTIELIAYNKIGNSSISSVSFIAASKA
cDNA Sequences
>Archaeoglobus
TABLE-US-00020 [0397] SEQ ID NO: 89
gccatggcagccCCGGCGATTAGCAATGTGCGCGTTAGCGATGTGACC
AACAGCAGCGCCACCATTCGTTGGGATGTGAGCCTGGCGGCGAAT
AATCGCGTGCTGTTTAGCACCAACAGCGATCTGAGCAGCCCGCAG
TGGAGCGCGTGGGATAACAGCACCGATAGCCCGATGATTACCCTG
AGCGGTCTGAGCGCGGGCACCGCGTATTATTTTAGCGTGTATAGCT
TTCGTCCGGATAATGCGAGCCTGTATAGCAACAGCAGCATTATGAG
CTTTACCACCggtggcggtacc SEQ ID NO: 90
CCGGCGATTAGCAATGTGCGCGTTAGCGATGTGACCAACAGCAGCG
CCACCATTCGTTGGGATGTGAGCCTGGCGGCGAATAATCGCGTGCTG
TTTAGCACCAACAGCGATCTGAGCAGCCCGCAGTGGAGCGCGTGGG
ATAACAGCACCGATAGCCCGATGATTACCCTGAGCGGTCTGAGCGCG
GGCACCGCGTATTATTTTAGCGTGTATAGCTTTCGTCCGGATAATGCGA
GCCTGTATAGCAACAGCAGCATTATGAGCTTTACCACC
>Staphylothermus
TABLE-US-00021 [0398] SEQ ID NO: 91
gccatggccgccAGCGAACCGCAGAACCTGAAAGCGACCGCGGGTAATA
ACAATATTACCCTGACCTGGGATCCGCCGATTGATGATGGTGGCTGCCG
CATTGTGGAATATCGTATTTATCGTGGCACCAATAATAACAACCTGGAA
TATTATGCGAGCGTTAACGGCAGCACCACCACCTTTATTGATAAAAATA
TTGTGTATAGCCAGACCTATTATTATAAAGTGAGCGCGGTGAACAATAT
TGTGGAAGGCCCGAAAAGCAACACCGCGAGCGCGACCCCGACCAGCAGC ggtggcggtacc SEQ
ID NO: 92 AGCGAACCGCAGAACCTGAAAGCGACCGCGGGTAATAACAATATTACCC
TGACCTGGGATCCGCCGATTGATGATGGTGGCTGCCGCATTGTGGAATA
TCGTATTTATCGTGGCACCAATAATAACAACCTGGAATATTATGCGAGC
GTTAACGGCAGCACCACCACCTTTATTGATAAAAATATTGTGTATAGCC
AGACCTATTATTATAAAGTGAGCGCGGTGAACAATATTGTGGAAGGCCC
GAAAAGCAACACCGCGAGCGCGACCCCGACCAGCAGC
>S. solfataricus 1
TABLE-US-00022 SEQ ID NO: 93
gccatggccgccCCGCTCCCACCGAAAATTACCAGCTATAGCGCGGGC
AACGAAAGCGTGACCCTGGGCTGGAACCCGGTGCGTCTGAGCAGC
GGCTATGAAATTATTTATTGGAACAATATGGGCTTTAACAGCAGCATT
AATGTGGGTAATGTGACCAGCTATACCGTGACCGGCCTGAAAGATG
GCATTACCTATTATTTTGAAGTGCTGGCCTATAACAGCATTGGTTATA
GCAGCCCGAGCAGCATTATCGCGCTGACCCCGGCGAGCGTGggtgg cggtacc SEQ ID NO:
94 CCGCTCCCACCGAAAATTACCAGCTATAGCGCGGGCAACGAAAGC
GTGACCCTGGGCTGGAACCCGGTGCGTCTGAGCAGCGGCTATGAAA
TTATTTATTGGAACAATATGGGCTTTAACAGCAGCATTAATGTGGGTAA
TGTGACCAGCTATACCGTGACCGGCCTGAAAGATGGCATTACCTATTA
TTTTGAAGTGCTGGCCTATAACAGCATTGGTTATAGCAGCCCGAGCAG
CATTATCGCGCTGACCCCGGCGAGCGTG
>S. tokodaii 1
TABLE-US-00023 SEQ ID NO: 95
gccatggccgccCCGCCGAAACCGCAGATTGCCAGCATTGCCAGCGGT
AATGAAACCATTACCGTGAAATGGTATGATACCAATGCGAGCGGCT
ATTATATTACCTATTGGAGCAATTTTAGCCAGAAAGTGACCATTAATG
TGGGTAACGTGACCAGCTATACCATTAAACATCTGAAAGATGGCGTG
ACCTATTATATTCAGATTGTGCCGTATAACAGCCTGGGCAATGGCAC
CCCGAGCGATATTATTAGCGCGACCCCGAGCAGCGTTggtggcggtacc SEQ ID NO: 96
CCGCCGAAACCGCAGATTGCCAGCATTGCCAGCGGTAATGAAACCA
TTACCGTGAAATGGTATGATACCAATGCGAGCGGCTATTATATTACCTA
TTGGAGCAATTTTAGCCAGAAAGTGACCATTAATGTGGGTAACGTGAC
CAGCTATACCATTAAACATCTGAAAGATGGCGTGACCTATTATATTCAG
ATTGTGCCGTATAACAGCCTGGGCAATGGCACCCCGAGCGATATTATT
AGCGCGACCCCGAGCAGCGTT
>S. tokodaii.sub.--2
TABLE-US-00024 SEQ ID NO: 97
gccatggccgccCCGAATCCGCCGATTATTAAAGTGAAAATTGGCAATC
TGAATGCGACCCTGACCTGGTATGATACCTTTAATGGTGGTTATCCGAT
TGAAGGCTATTATCTGTATGTGAACGGTAAAGGTATTAACGTGGGCAAC
ATTACCAGCTATGTGCTGACCAATCTGACCGCCGGTGAACTGTATACCA
TTGAACTGATTGCGTATAACAAAATCGGCAACAGCAGCATTAGCAGCGT
GAGCTTTATTGCGGCGAGCAAAGCGggtggcggtacc SEQ ID NO: 98
CCGAATCCGCCGATTATTAAAGTGAAAATTGGCAATCTGAATGCGACCC
TGACCTGGTATGATACCTTTAATGGTGGTTATCCGATTGAAGGCTATTA
TCTGTATGTGAACGGTAAAGGTATTAACGTGGGCAACATTACCAGCTAT
GTGCTGACCAATCTGACCGCCGGTGAACTGTATACCATTGAACTGATTG
CGTATAACAAAATCGGCAACAGCAGCATTAGCAGCGTGAGCTTTATTGC GGCGAGCAAAGCG
Vector Sequence
TABLE-US-00025 [0399] SEQ ID NO: 99
GACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAAT
GGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATT
TGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTG
ATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGT
GTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAA
ACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCTCGAGTGGGTTAC
ATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC
GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGT
ATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT
TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAA
GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACT
TCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGG
GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC
AAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAA
ACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGG
ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCT
GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGC
AGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG
GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC
ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT
GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA
TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC
GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT
GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG
AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA
TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCA
CCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG
ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC
AGCGGTCGGGCTGAACGGGGGGTTCGTGCATACAGCCCAGCTTGGAGCGAACGA
CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC
CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA
GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC
GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC
GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG
CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACC
GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG
CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCT
CCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGG
AAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCA
CCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTTGGAG
CCTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTT
GTTCCTTTCTATGCGGCCCAGCCGGCCATGGCCGCCATTGAAGTGAAAGATGTGA
CCGATACCACCGCGCTGATTACCTGGTTTAAACCGCTGGCGGAAATTGATGGCTG
TGAACTGACCTATGGCATTAAAGATGTGCCGGGCGATCGCACCACCATAGATCTG
ACCGAAGATGAAAACCAGTATAGCATTGGTAACCTGAAACCGGATACCGAATAT
GAAGTGAGCCTGATTTGCCGTCGTGGCGATATGAGCGGCGCGCCGGCGAAAGAA
ACCTTTACCACCGGTGGCGGTACCCCAACCGACCCGCCAACCACTCCACCAACTG
ATAGCCCAGGCGGTACTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGAT
GGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACA
GTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATC
GATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTG
ATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACC
TTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTC
GCCCTTTTGTCTTTAGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAA
ATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTAT
GTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAAGAATTCGACG
GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCG
TCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAG
ATTGTACTGAGACTGCACCATAAAATTGTAAACGTTAATATTTTGTTAAAATTCG
CGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAA
AATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGTTGTTCCAGTT
TGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAA
ACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCAAATCAAGTTTTT
TGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGAT
TTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAA
GCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTA
ACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTACTATGGTTGCT
TTGACGTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCAT
CAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCG
GGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT
AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAG
TTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGA
TABLE-US-00026 Seq ID Clone BC loop Seq ID NO: FG loop NO: SYNAGIS
.RTM. specific binding scaffolds SynBP01 SPPSVLVGYTG 100 VTEFGRRRS
101 2 FKPLAEIDG 102 RKIIGLLSNPA 103 3 FKPLAEIDG 102 GTVVGQKSNPA 104
4 SPGERIWMFTG 105 PNYERISNPA 106 5 SPSGRVILWTG 107 DNLYGRISNPA 108
6 ATPGCRNGKIVG 109 TTSVGATSNPA 110 Lysozyme specific binding
scaffolds 1 FKPLAEIDG 111 YNRYGLCPS 112 2 FKPLAEIDG 111 SNRIGMCPS
113 3 AQPTSPNGSIXG 114 RRGDMSSNPA 115 4 FKPLAEIDG 102 DTVHGRLSNPA
116 5 FKPLAEIDG 102 RKVLGRLSNPA 117 6 FKPLAEIDG 102 RKLVGALRS 118 7
FKPLAEIDG 102 RKVLRYSNPA 119 8 SPCNGGKRCTG 120 RRGDMSSNPA 121 9
FKPLAEIDG 102 FKWLGAIRS 122 10 FKPLAEIDG 102 GNCVGNLWS 123 11
SPAWITWHRTG 124 HTPLGHLRS 125 TRAIL-R2 two-loop binders Clone SEQ
ID name BC loop SEQ ID NO. FG loop NO. 2F4 SPCIMVCLRTG 126
RRGDMSGAPA 127 5B10 SPCLFVCLRTG 128 RRGDMSGAPA 129 10D9 SPPLFCCQKTG
130 FKLTGFLYS 131 6F11 SPSVARMLETG 132 ITLCGRGVS 133 8B3
SPPEYAFYYTG 134 VKNCGLFSNPA 135 5E5 SLAPGYRLG 136 VKLCMRGNPA 137
2H6 ATPSVFDSHIEG 138 WKHHGDAWS 139 7G11 AKPSIVNGFISG 140 DKCFGAMKS
141 6C7 AKPMSCSGYIQG 142 AKLTGWLCS 143 TRAIL-R2 three-loop binders
Seq Seq Seq Clone BC loop Id. DE loop Id. FG loop Id. 1E03
AAPFFGSSYISG 144 HYYVTR 145 VNLSGHMPS 146 2B04 APPMLTDSEING 147
TSSYWS 148 STLRRNAIS 149 1C12 AKPEKWDGSIYG 150 NSRHTA 151
FTPYGAKSNPA 152 1A03 APPPFSNSCIIG 153 RPGRAS 154 STGTGLPSNPA 155
1C10 SPCCPYDRYTG 156 QSSRSH 157 ITTFGHVSNPA 158 1B12 AKPRQGGSNISG
159 YHKGLH 160 PKMTGYTYS 161 2G03 SPGPLLRHTTG 162 RPIPRA 163
RNRPQQSNPA 164 2D3 SPGGFQKITTG 165 VNRRNH 166 LTYKARAIS 167 1C06
SPRMYTWIQTG 168 THLSGS 169 LKLTRTHIS 170 2F08 SHAGGIRIG 171 HVWQVY
172 MTPYLLGNPA 173 1B04 SPSHGVESSTG 174 HGLQRV 175 AKICGHLVS 176
3B11 SPCQLLALITG 177 NSRHYH 178 YTSTGQRSNPA 179 1D8 SPCQMLSSLTG 180
NIERPK 181 FTMTGYRSNPA 182 2A12 SPCCQEFTLTG 183 HNHHHH 184
ITDAGNKSNPA 185 1E05 SPCSPCQLVTG 186 SCTRAK 187 INKLGDTSNPA 188
2F02 SPSRGGTSLTG 189 DQVRAT 190 HTNSGQPSNPA 191 1H05 SPGMFDQVRTG
192 GKYWER 193 RNQYGQHQS 194 2A11 SPPFRAGHVTG 195 VTARCQ 196
TTGNGLRSNPA 197 1G11 SWAQANPGG 198 WHSITF 199 KTKVQSSNPA 200
8. EXAMPLES
[0400] The invention is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these examples but rather should be construed
to encompass any and all variations which become evident as a
result of the teachings provided herein.
8.1 Example 1
Recombinant Expression of Candidate Scaffolds
[0401] This example demonstrates that candidate scaffolds may be
recombinantly expressed in E. coli in sufficient quantity to be
visible against the background of host proteins on a Coomassie
stained on a polyacrylamide gel (FIG. 1). Represented in FIG. 1 is
the PAGE analysis of crude E. coli lysates expressing candidate
scaffolds. Some of the candidate scaffolds exhibited higher
expression levels (exemplified by lanes 3, 4, 5, and 10) and were
selected for further development. Specifically, Lane 3 represents
the 6.sup.th domain of Fibronectin III, Lane 4 represents the
scaffold from .beta.-common receptor, Lane 5 represents the
candidate scaffold from growth hormone receptor and Lane 10
represents the Tn3 structural motif.
[0402] Recombinant Expression
[0403] A panel of human-derived Tn3 structural motif sequences were
selected for heterologous expression in E. coli (Table 1).
Synthetic cDNAs encoding each of these proteins, and optimized for
codon usage in E. coli, were supplied by GenScript Corporation as
per the sequences shown. Each cDNA contained flanking Nco I and Kpn
I restriction sites, and following digestion with these enzymes,
the inserts were cloned into a modified pET22b vector (Novagen)
containing corresponding Nco I/Kpn I sites.
[0404] In other embodiments, vectors may comprise any number of
restrictions sites to facilitate the engineering of the scaffolds
of the invention. In some embodiments, vectors of the invention
comprise at least one restriction site. In other embodiments,
vectors of the invention comprise at least one restriction site
flanking at least one loop sequence. In other embodiments, vectors
of the invention comprise at least one restriction site selected
from the group consisting of NcoI, BgIII, BstEII, AscI, and KpnI.
In further embodiments, the vector comprises a leader sequence. In
other embodiments, the vector comprises a linker sequence. In a
specific embodiment, vectors of the invention comprise the
polynucleotide sequence defined by SEQ ID NO:99.
[0405] Transformants of BL21 DE3 E. coli harboring Tn3 structural
motif expression plasmids were grown overnight at 37.degree. C. in
Luria Broth containing 50 .mu.g/mL carbenicillum. Overnight
cultures were diluted 1 in 20 into Super Broth media containing 50
.mu.g/mL carbenicillum and 2% w/v glucose and incubated at
37.degree. C. with shaking until the optical density at 600 nm was
0.6. At this time, protein expression was induced by addition of
IPTG to 200 .mu.M, and cultures were transferred to a 30.degree. C.
incubator with shaking. After 5 h at 30.degree. C., a small aliquot
of culture was removed for SDS-PAGE analysis, and the remainder of
cells were pelleted by centrifugation, and frozen overnight at
-20.degree. C. SDS-PAGE analysis of whole cell lysates suggested
that Tn3 structural motifs 3, 4, 5, 10 were highly overexpressed in
E. coli as determined by gel bands at approximately 10 kDa (FIG.
1). In a separate experiment, it was also determined that Tn3
structural motif 12 was overexpressed.
[0406] Purification
[0407] Frozen cell pellets were resuspended in lysis buffer (50 mM
NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH 8.0) containing
1 mg/mL lysozyme (Sigma) and 200 units/mL of DNase (Invitrogen).
Lysis was effected by sonication, and clarified lysate was
separated from cell debris by centrifugation followed by filtration
through a 0.8 .mu.m filter. Lysates were loaded onto HiTrap
chelating columns charged with Ni.sup.2+, washed with 15 column
volumes of wash buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM
imidazole, pH 8.0) and eluted with 4 column volumes of elution
buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250 mM imidazole, pH
8.0). The concentrations of purified protein were determined by UV
absorbance at 280 nm according to Gill and von Hippel (Anal.
Biochem. 182: 319, 1989). Post purification yields of the various
Tn3 proteins are reported in Table 1. The Tn3 structural motif
derived from human tenascin C (Tn3) gave the highest yield, which
corresponded to 110 mg of purified Tn3 obtained from a 400 mL
culture. SDS-PAGE analysis of a purified Tn3 sample is shown in
FIG. 2.
TABLE-US-00027 TABLE 1 Protein Expression Protein DNA # Parent
Protein level (mg/L) sequence sequence 1 Fibronectin 2 mg/ml 1P 1D
2 Fibronectin -- 2P 2D 3 Fibronectin 83 3P 3D 4 Growth hormone R 48
4P 4D 5 .beta.-common R 9 5P 5D 6 IL-5R -- 6P 6D 7 Tenascin XB --
7P 7D 8 Tenascin XB -- 8P 8D 9 Tenascin XB -- 9P 9D 10 Tenascin C
(Tn3) 265 10P 10D 11 Growth hormone R -- 11P 11D 12 PTPR-F 105 12P
12D 13 PTPR-F 3 13P 13D 14 Collagen type XIV 1 14P 14D
[0408] Characterization
[0409] Given the high yield of soluble Tn3 produced in E. coli,
this protein was analysed for its stability and solution
properties.
[0410] Stability
[0411] Thermal unfolding of Tn3 was assessed by differential
scanning calorimetry (DSC). A 1 mg/mL Tn3 sample in 20 mM sodium
phosphate at pH 7.0 exhibited a melting temperature (Tm) of
45.degree. C. (FIG. 3A), moreover, thermal unfolding was reversible
as evidenced by superimposable thermograms when the same sample was
cooled and reheated. Tn3 was more stable to thermal unfolding at
lower pH or high salt. The Tm in 20 mM sodium acetate pH 5.0 was
56.degree. C., and 55.degree. C. in 20 mM sodium phosphate pH 7.0
containing 1 M NaCl.
[0412] Unfolding of Tn3 by chaotropic agents was monitored by
intrinsic fluorescence. Samples of 0.1 mg/mL Tn3 containing
different concentrations of urea or guanidine hydrochloride were
prepared in 20 mM sodium phosphate pH 7.0., or 20 mM Tris pH 7.5.
Fluorescence emission spectra were acquired on a Photon Technology
QuantaMaster spectrofluorometer at an excitation wavelength of 280
nm. In the absence of chaotrope, folded samples of Tn3 exhibited an
emission maxima at 319 nm. Unfolding of Tn3 by urea or GuHCl
resulted in a red shift of the maxima to 348 nm, in addition to an
increase in fluorescence intensity. The midpoint of unfolding at pH
7.0 or 7.5 occurred at approximately 2M urea (FIG. 3B) or 0.8M
GuHCl.
[0413] The stability of Tn3 to proteolytic degradation was tested
by incubation with thermolysin. Tn3 (45 .mu.M) in digest buffer (20
mM Tris pH 7.5 containing 10 mM CaCl.sub.2) was incubated at room
temperature with thermolysin (0.45 .mu.M). Aliquots of the digest
were removed at different time points, and the reaction quenched by
addition of excess EDTA. Samples were then analysed by SDS-PAGE
(see the WT lanes in FIG. 10G). As demonstrated by FIG. 10G, the
wild-type Tn3 domain is rapidly degraded when incubated with
thermolysin.
[0414] Size exclusion chromatography with multi-angle light
scattering (SEC-MALS) was used to determine whether Tn3 was
monomeric in solution. Size exclusion separation was carried out
using a Bio-Rad Bio-Sil SEC 125-5 column (7.8.times.300 mm) at a
flow rate of 0.75 mL/min. The mobile phase was phosphate-buffered
saline (PBS) at pH7.2. Triple detection was accomplished using a
Wyatt Technologies DAWN EOS multi-angle light scattering detector
coupled with a Wyatt Technologies Optilab rEX differential
refractive index detector and an Agilent 1100 Series variable
wavelength UV detector. SEC-MALS analysis showed that the monomer
content of a 4.5 mg/mL Tn3 stored at 4.degree. C. for 2 months was
97% (FIG. 3C). The experimentally-derived monomer mass was 10.6 kDa
which is in close agreement with the calculated mass of 10.8 kDa.
The Tn3 scaffold remains as a monomer as a monomer even through
extended periods of storage.
8.2 Example 2
Identification of Loop Length and Sequence Diversity
[0415] An ideal scaffold is highly soluble and stable. It should be
small enough for structural analysis, yet large enough to
accommodate a multitude of changes to facilitate binding of a
target. In an effort to facilitate the identification and
engineering of non-antibody protein scaffolds as well as design of
combinatorial libraries of scaffolds, a bioinformatics analysis of
scaffold sequences was performed.
[0416] The Tn3 structural motif is small, monomeric, soluble and
stable. In addition, Tn3 structural motifs are present in many
different human proteins, providing important information on
conserved residues which are often important for the stability and
folding as well as regions of diversity which can be exploited to
introduce novel binding functions. From sequence analysis, large
variations are seen in the BC and FG loops, suggesting that the
loops are not crucial to stability. Using this property, a strategy
was developed to identify candidate protein scaffolds and analyze
the loop length and sequence diversity in an effort to characterize
the natural extent of variation that occurs in these two
parameters.
[0417] A search of the available protein databases identified a
number of protein scaffolds based on the Tn3 structural motif.
Candidate scaffolds contained a similar predicted structure to Tn3
structural motifs, namely 7 beta strands each separated by a loop
region. An analysis of the location of the beta strands and the
loop regions revealed a pattern of diversity that may aide in the
prediction of loop length and sequence compositions for candidate
scaffolds for which a structure is not available. A length
diversity analysis was performed for the BC, DE, and FG loops of
candidate scaffolds in accessible protein databases.
[0418] A compilation of identified scaffold sequences has lead to
the development of the loop length diversity graphs presented in
FIGS. 4, 5, and 6. Presented in FIG. 4A is the BC loop length
diversity obtained from the analysis of 51 sequences from the
Protein Data Bank (PDB). It is apparent from the graph that the BC
loop length in this collection of sequences ranges from about 8
amino acid residues to about 26 amino acid residues, with the 9
amino acid residue loop being the most predominant. Presented in
FIG. 4B is loop length diversity obtained from the analysis of 397
sequences identified from the Swiss-prot database. It is apparent
from the graph that the BC loop length in this collection of
sequences ranges from about 7 amino acid residues to about 19 amino
acid residues, with the 12 amino acid residue loop being the most
predominant. Presented in FIG. 5 is the FG loop length diversity
obtained from the analysis of 51 sequences identified from the PDB
database. It is apparent from the graph that the FG loop length in
this collection of sequences ranges from about 6 amino acid
residues to about 18 amino acid residues, with the 10 amino acid
residue loop being the most predominant. A similar analysis as
described herein was performed for the DE loop sequences. Presented
in FIG. 6 is the DE loop length diversity, demonstrating that the
length of DE loops range from about 4 amino acid residues to about
17 amino acid residues with the 6 residue loop being the most
predominant length.
[0419] In addition to the length diversity, for the more abundant
loop lengths a sequence diversity analysis was performed in an
attempt to guide the establishment of consensus sequences.
Presented in FIG. 7A is the sequence diversity graph of 9 amino
acid residue BC loops. The prevalence of a particular amino acid at
each position is represented by the relative size of the box
containing that residue over each position. For example, at
position 3 of a 9 amino acid residue loop, there is a preference of
a proline or an alanine. Also, at position 5 there is a preference
for glycine and alanine. At position 7 there is a preference for
the amino acids valine, isoleucine, and phenylalanine. Other
positions in the loop show greater sequence diversity suggesting
that these positions may be suitable for complete randomization in
construction of libraries of the Tn3 scaffold. Presented in FIG. 7B
is the sequence diversity graph of 12 amino acid residue BC loops.
In this analysis, for example, at position 3 a Proline residue is
preferred. Also, positions 1, 4, 5 and 12 appear to be suitable for
complete randomization as they do not exhibit selectivity for an
amino acid residue. Presented in FIG. 8 is the sequence diversity
graph for all lengths of FG loops. From this analysis, position 2
is often asparagine, position 5 prefers glycine, position 7 is
often glycine or serine. Also, positions 1, 3, 4, 6, 8, 9, 11, 12,
and 13 represent candidate positions for complete randomization in
library construction as they do not demonstrate selectivity for
amino acid residues. The data from this analysis suggests a
potential benefit of limiting loop diversity at positions showing a
sequence conservation such that a greater proportion of molecules
in a library may maintain WT-like stability, expression and
solubility.
8.3 Example 3
Construction of a Phage Displayed Tn3 Library and Selection of
Specific Binders
[0420] Given the loop and sequence diversity established above, a
directed approach to the development of libraries was performed.
More specifically, loops BC and FG were randomized in a restricted
fashion. The following strategy was employed for the BC and FG
loops.
[0421] Library Design and Construction
[0422] A synthetic cDNA encoding Tn3, corresponding to the Nco
I-Kpn I fragment of seq. ID 10D (See section 6 for sequence), was
cloned into a phage display vector enabling display of Tn3 protein
on the surface of M13 bacteriophage as a fusion to a fragment of
the gene III coat protein. The construct encoded a 20 amino acid
Thr/Pro-rich linker sequence between the C-terminus of Tn3 and
codons 251-406 of M13 gene III.
[0423] Libraries of randomly mutated Tn3 were prepared by Kunkel
mutagenesis (Kunkel T A et al., Methods Enzym. 204, 125, 1991)
using degenerate oligonucleotides. Three degenerate
oligonucleotides were used to randomize the coding sequence of the
BC loop, and three for the FG loop (Table 2). This strategy
resulted in the introduction of characterized sequence and loop
length diversity into the Tn3 library, consistent with patterns of
diversity described for natural Tn3 domains.
TABLE-US-00028 TABLE 2 Degenerate oligonucleotides for Tn3 library
construction Oligo Loop name randomized Sequence Seq ID BC9 BC
ACCGCGCTGATTACCTGGTCTNNK 211 SCGNNKGSTNNKNNKNNKGGCATTG
AACTGACCTATGGC BC11 BC ACCGCGCTGATTACCTGGTCTCCG 212
BSTNNKNNKNNKNNKNNKNNKACCG GCATTGAACTGACCTATGGC BC12 BC
ACCGCGCTGATTACCTGGGCGVMA 213 CCGNNKNNKNNKRRCRGCNNKATTNN
KGGTATTGAACTGACCTATGGC FG9 FG TATGAAGTGAGCCTGATTAGCNNK 214
AMSNNKNNKGGTNNKNNKNNKAGCA AAGAAACCTTTACCACC FG10 FG
TATGAAGTGAGCCTGATTAGCNNK 215 AMSNNKNNKNNKNNKRGCAACCCGG
CGAAAGAAACCTTTACCACC FG11 FG TATGAAGTGAGCCTGATTAGCNNK 216
AMSNNKNNKGGTNNKNNKAGCAAC CCGGCGAAAGAAACCTTTACCACC
[0424] Following transformation of electrocompetent E. coli with
randomly mutated phagemid constructs, M13KO7 helper phage was added
and cultures were grown overnight at 37.degree. C. in 500 mL of 2YT
medium containing carbenicillum. Phage was then isolated from
culture supernatants by precipitation with a saline polyethylene
glycol solution and resuspended in a small volume of PBS.
[0425] Panning of Libraries for a Specific SYNAGIS.RTM. Binding
Scaffold
[0426] SYNAGIS.RTM. was passively adsorbed onto microtitre plate
wells, and free sites were blocked with PBS containing 10 mg/mL BSA
or casein. Phage stocks were diluted with PBS containing 2 mg/mL
BSA or casein and 0.1% v/v Tween-20. Diluted phage samples (100
.mu.L; .about.10.sup.12 phage) were added to SYNAGIS.RTM.-coated
wells and incubated for 2 h at room temperature with gentle
shaking. Plates were then washed 10-15 times with PBS containing
0.1% v/v Tween 20, and bound phage were eluted with 100 .mu.L/well
of 0.1M HCl, then neutralized by addition of 1.0M Tris-HCl, pH 8.0.
Eluted phage were re-propagated by infection of XL-1 Blue E. coli
and harvested from overnight cultures co-infected with M13KO7
helper phage in 50 mL 2YT medium containing 50 .mu.g/mL
carbenicillum. The library was panned against SYNAGIS.RTM. in this
manner for a total of four rounds, using BSA in the blocking and
diluent buffers in the first and third rounds, and casein in rounds
2 and 4.
[0427] Screening of SYNAGIS.RTM. Binding Clones
[0428] After the final round of panning, eluted phage were serially
diluted and used to infect XL-1 Blue E. coli for 1 h prior to
plating overnight on LB agar containing 50 .mu.g/mL carbenicillum.
Individual colonies were picked and grown overnight in
2YT/carbenicillum at 37.degree. C. Cultures were then diluted 1:100
in 2YT/carbenicillum, grown to an optical density (600 nm) of 0.4,
and M13KO7 helper phage were added followed by overnight incubation
at 37.degree. C. with shaking. Following centrifugation, 50 .mu.L
of culture supernatant was diluted with an equal volume of PBS
containing 0.1% v/v Tween 20 and 2% w/v skim milk powder, and used
for analysis by ELISA. Clones that gave the highest response for
binding to SYNAGIS.RTM. but not BSA/casein-coated ELISA plates were
then selected for sequencing. To determine the sequences of Tn3
variants displayed by the selected clones, 1 .mu.L of culture
supernatant was used in a PCR to amplify a fragment encompassing
the encoded Tn3 sequence. This PCR product was then treated with
ExoSAPit, (USB Corp., Cleveland, Ohio) to degrade unconsumed
deoxynucleotides and primers, and sequenced directly.
[0429] Identification of the SYNAGIS.RTM.-Binding Tn3 Variant
SynBP01
[0430] DNA sequencing of SYNAGIS.RTM.-binding Tn3 variants
identified 3 unique clones. The clone with the highest signal in a
SYNAGIS.RTM.-binding ELISA, SynBP01, contained novel BC and FG loop
sequences as shown in Table 3. To express soluble SynBP01, the
NcoI-KpnI cDNA fragment was excised from the phage display vector
and cloned into the corresponding sites of the E. coli expression
vector previously described. Recombinant SynBP01 was then expressed
in E. coli, and purified as previously described for wild type Tn3.
The yield of purified SynBP01 was 18 mg from 400 mL of E. coli
culture, and was greater than 95% pure as judged by SDS-PAGE
analysis (FIG. 9A). As this recovery is close to the capacity of
the 1 mL nickel chelate affinity column used, it is likely that
this yield under represents the actual expression level of
SynBP01.
[0431] Determination of SYNAGIS.RTM. Binding Affinity Exhibited by
SynBP01
[0432] The equilibrium dissociation constant (K.sub.D) for binding
of SynBP01 to SYNAGIS.RTM. was measured by surface plasmon
resonance on a BIAcore 3000 instrument. SYNAGIS.RTM. was covalently
immobilized on the biosensor chip via primary amino groups.
Equilibrium binding of SynBP01 was measured by injecting samples of
SynBP01 in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH
7.5, 0.005% v/v Tween 20) over the chip at a flow rate of 10
.mu.L/min (FIG. 9B). Following each equilibrium binding
measurement, SynBP01 was allowed to completely dissociate by
running HBS-EP buffer over the chip for at least 25 mM prior to
injection of the next SynBP01 sample. Binding profiles were
analyzed by using BIAevaluation software (Biacore AB, Uppsala,
Sweden), and the K.sub.D for binding of SynBP01 to SYNAGIS.RTM. was
calculated as 16 .mu.M.
[0433] Examination of the Stability of SynBP01
[0434] The stability of SynBP01 to unfolding by urea at pH 7.0 was
measured as described previously for Tn3 (See Example 1). To
facilitate a comparison of chaotrope-induced unfolding of Tn3 and
SynBP01, the relative fluorescence emission intensity at 360 nm was
plotted as a function of chaotrope concentration for each
protein.
[0435] A comparison of urea-induced unfolding of Tn3 and SynBP01 at
pH 7.0 (FIG. 9C) showed that the concentration of urea required to
achieve 50% unfolding was similar for both proteins. This indicates
that the stability of SynBP01 is similar to that of wild type
Tn3.
[0436] In other library screens, additional SYNAGIS.RTM. and
Lysozyme specific binding scaffolds were identified. The specific
BC and FG loops sequences are as follows:
TABLE-US-00029 TABLE 3 BC and FG loop sequences of SYNAGIS .RTM.
and Lysozyme specific binding scaffolds Seq ID Clone BC loop Seq ID
NO: FG loop NO: SYNAGIS .RTM. specific binding scaffolds SynBP01
SPPSVLVGYTG 100 VTEFGRRRS 101 2 FKPLAEIDG 102 RKIIGLLSNPA 103 3
FKPLAEIDG 102 GTVVGQKSNPA 104 Lysozyme specific binding scaffolds 1
FKPLAEIDG 102 YNRYGLCPS 112 2 FKPLAEIDG 102 SNRIGMCPS 113 3
AQPTSPNGSIXG 114 RRGDMSSNPA 115 4 FKPLAEIDG 102 DTVHGRLSNPA 116 5
FKPLAEIDG 102 RKVLGRLSNPA 117 6 FKPLAEIDG 102 RKLVGALRS 118 7
FKPLAEIDG 102 RKVLRYSNPA 119 8 SPCNGGKRCTG 120 RRGDMSSNPA 121 9
FKPLAEIDG 102 FKWLGAIRS 122 10 FKPLAEIDG 102 GNCVGNLWS 123 11
SPAWITWHRTG 124 HTPLGHLRS 125
[0437] mAb Capture of Paired Scaffolds
[0438] Bivalent or bispecific scaffolds, containing 2 identical or
2 distinct scaffolds, may be useful therapeutic molecules based on
their ability to simultaneously bind 2 target proteins. A method of
directly screening pairs of scaffolds for a desired bivalent or
bispecific activity, without having to reformat and express tandem
or fusion scaffolds, would vastly simply the process of identifying
useful scaffold pairs. Only those pairs identified in the screen
would then require reformatting into a form suitable for
preparation of a bivalent or bispecific scaffold (for example, a
multimeric scaffold).
[0439] Full length IgG antibody molecules contain two antigen
binding sites, and can therefore bind two target antigens
simultaneously. Thus, noncovalent monoclonal antibody capture of
scaffolds would result in presentation of two scaffolds on each
antigen-binding arm of the antibody. If the scaffold captured in
this way were oriented so that the binding surface formed by the
BC, DE and FG loops were available for binding to other proteins,
then this could form the basis for screening pairs of scaffolds.
For this reason, capture by an antibody which recognizes an epitope
involving the C-terminus of the scaffold, or any/all of the AB, CD,
EF loops would be most suitable. Antibody capture of 2 identical
scaffolds would result in formation of a homodimeric mAb-scaffold
complex that could mimic the activity of a bivalent scaffold.
Capture of 2 different scaffolds would result in a mixture of homo-
and heterodimeric mAb-scaffold complexes, wherein the heterodimeric
complex could mimic the activity of a bispecific scaffold.
[0440] To demonstrate the feasibility of this assay strategy,
different concentrations of PentaHis, an anti-histidine tag
monoclonal antibody (Qiagen), was incubated for 2 h with 1 .mu.M
SynBP01 in HBS-EP buffer. PentaHis capture of SynBP01 was not
expected to interfere with its ability to bind SYNAGIS.RTM., given
the C-terminal hexahistidine tag should be on the opposite side of
the molecule to the binding surface formed by the BC, DE and FG
loops (FIG. 9D). The PentaHis-SynBP01 samples were then injected
over a BIAcore.RTM. chip onto which SYNAGIS.RTM. had been
immobilized as previously described. The sensorgrams were corrected
for background by subtraction of corresponding sensorgrams for
injection of PentaHis in the absence of SynBP01, and then compared
to the sensorgram obtained for injection of 1 .mu.M SynBP01 in the
absence of PentaHis.
[0441] Binding of the PentaHis-SynBP01 complex to SYNAGIS.RTM. was
significantly stronger than binding of free SynBP01, as exemplified
by the sensorgram corresponding to the complex of 0.19 .mu.M
PentaHis with 1 .mu.M SynBP01 (FIG. 9D) In particular,
PentaHis-complexed SynBP01 had a much slower dissociation rate from
the SYNAGIS.RTM. surface, indicative of bivalent binding, than did
free SynBP01. Control experiments verified that PentaHis did not
exhibit any detectable binding to SynBP01 when injected alone, nor
did injection of a SynBP01/irrelevant mouse mAb mixture (isotype
matched with PentaHis) show any evidence of enhanced binding
relative to SynBP01 alone.
8.4 Example 4
Design, Expression and Characterization of Disulfide-stabilized Tn3
Variants
[0442] Design of Disulfide-Containing Tn3 Variants
[0443] While many naturally occurring Tn3 structural motifs lack
disulfide bonds, others do contain one or more disulfide bonds.
Thus, rather than attempt de novo design of disulfide bonds in Tn3
by measuring distances and angles between amino acid side chains,
we introduced cysteine residues into the Tn3 scaffold at positions
analogous to Tn3 structural domains that contain naturally
occurring disulfides.
[0444] The three dimensional structures of 21 Tn3 structural motifs
that naturally contain disulfide bonds were superimposed. The PDB
codes and sequences for these structures is shown in FIG. 10A.
Within these 21 structures, disulfide bonds occurred at various
positions within the scaffold, however, a number of these were
analogous in that the disulfide bonds were overlaid within the
family of superimposed structures. Three examples were found of
disulfide bonds that were represented more than twice across the 21
structures. Two of these three correspond to the previously
described disulfide bond pair commonly conserved within cytokine
receptors (Bazan et al., PNAS, 87, 6934, 1990). A total of 8 and 12
instances of these two disulfides were noted in the 21 structures.
The third disulfide occurs less frequently (5 cases/21 structures),
and results in a crosslink between the F- and G-strands.
[0445] Three cysteine-containing Tn3 mutants were designed in an
effort to introduce disulfide bonds at positions corresponding to
these 3 naturally occurring disulfides. A fourth
cysteine-containing mutant was also designed to introduce a
disulfide which occurs naturally in one of the Tn3 structural
motifs from mouse G-CSF receptor (PDB code 1 pgr). Although there
was only one instance of this disulfide in the 21 structures, it
results in a disulfide cross-link between the C- and F-strands that
is buried within the protein core and occurs close to the BC and FG
loop. For each of the Tn3 mutants, designated Tn3.sup.SS1-4, 2
cysteines were introduced at positions determined from the
structure-based sequence alignment of Tn3 with those of the
disulfide-containing Tn3 proteins (FIG. 10A). As disulfide 2 occurs
across at the base of the DE loop in Tn3 structural motifs which
often have longer DE loops tha Tn3, 2 glycine residues were also
inserted into the mutant corresponding to Tn3.sup.SS2. The
disulfide engineering strategy is graphically depicted in FIG.
10B.
[0446] Disulfide Mutant Generation and Recombinant Expression
[0447] Expression constructs for Tn3.sup.SS1-4 were generated by
site-directed mutagenesis of the wild type expression construct
described in Example 1. Recombinant protein was expressed in E.
coli and purified by immobilized nickel chelate affinity
chromatography as described in Example 1. All Tn3 mutants expressed
at a level in excess of 50 mg/L.
[0448] Refolding of Tn3.sup.SS1-4
[0449] Cytoplasmic expression of proteins in E. coli generally
results in the isolation of cysteine-containing proteins in the
reduced state, or with some degree of inappropriate disulfide
formation. To determine whether recombinant Tn3.sup.SS1-4 proteins
were oxidized (i.e. disulfide-containing) or reduced (i.e. lacking
disulfide bonds), purified material was analyzed by reverse phase
HPLC, and compared to material that was first pre-incubated with
the strong reducing agent DTT in the presence of guanidine
hydrochloride. In all cases, DTT-treated material chromatographed
as a single peak, while untreated samples showed the presence of a
2.sup.nd minor peak indicative of partial oxidation to the
disulfide-containing form (Representative HPLC analyses of
Tn.sup.SS3 and Tn.sup.SS4 are depicted in FIG. 10C).
[0450] In order to effect complete conversion of reduced
Tn3.sup.SS1-4 to the corresponding disulfide-containing proteins,
samples were diluted to 1 mg/mL with 6M guanidine hydrochloride
containing 10 mM DTT and buffered at pH 8. After 10 minutes
incubation at room temperature, samples were dialyzed overnight
against 0.5M guanidine hydrochloride buffered at pH 8.5 with 20 mM
Tris-HCl. HPLC-analysis of material refolded in this way typically
showed 30-70% conversion into the disulfide-containing species
(FIG. 10C). Further conversion to 90-100% of the
disulfide-containing species was affected by overnight incubation
at 37.degree. C., or by storing samples at 4.degree. C. for >2
weeks. Alternatively, near quantitative refolding to the
disulfide-containing product could be affected by overnight
dialysis of reduced and denatured protein into buffer containing
0.5M guanidine hydrochloride, 4 mM reduced glutathione, 0.8 mM
oxidized glutathione and buffered at pH 8.5 with 20 mM
Tris-HCl.
[0451] Characterization
[0452] To determine purity and whether disulfide bonds within
refolded Tn3.sup.SS1-4 samples were inter- or intramolecular,
samples were analyzed by SDS-PAGE under reducing and non-reducing
conditions (FIG. 10D). In the presence of reducing agent, all
refolded Tn3.sup.SS1-4 samples migrated at a similar position to
Tn3. In the absence of reducing agent, Tn3.sup.SS1, Tn3.sup.SS3 and
Tn3.sup.SS4 migrated similarly to Tn3, and were therefore expected
to contain an intramolecular disulfide as designed. By contrast,
Tn3.sup.SS2 migrated primarily as a dimer, with a small amount of
protein migrating as a monomer. Accordingly, Tn3.sup.SS2 appears to
form intermolecular disulfide-linked dimers and was therefore not
studied further.
[0453] Stability
[0454] Unfolding of Tn3.sup.SS1,3,4 by chaotropic agents was
monitored by intrinsic fluorescence as previously described for
wild type Tn3. To facilitate a comparison of chaotrope-induced
unfolding of wild type and disulfide-containing Tn3, the relative
fluorescence emission intensity at 360 nm was plotted as a function
of chaotrope concentration for each protein.
[0455] A comparison of urea-induced unfolding of Tn3 and
Tn3.sup.SS1,3,4 at pH 7.0 (FIG. 10E) showed that Tn3.sup.SS1 was
less stable than the wild type protein, but that Tn3.sup.SS3 and
Tn3.sup.SS4 were considerably more stable. At pH 7.0, the
concentration of urea required to achieve 50% unfolding (Cm) of
wild type Tn3 was 2M. By contrast, the Cm for unfolding of
Tn3.sup.SS3 was 4M urea, while for Tn3.sup.SS4 Cm was at least 6M,
but could not be accurately determined as this protein was not
fully unfolded at the highest concentration of urea used in these
experiments (8M).
[0456] Unfolding of Tn3.sup.SS4 by guanidine hydrochloride (GuHCl)
at pH 5.0 was also determined by fluorescence and compared to
unfolding of wild type Tn3. Given GuHCl is a stronger denaturant
than urea, a complete unfolding transition was obtained by
analyzing the fluorescence of protein samples in concentrations of
GuHCl ranging from 0 to 5.5M (FIG. 10F). The Cm for unfolding of
wild type Tn3 at pH 5.0 was 1M GuHCl and approx. 3.2M for
Tn3.sup.SS4.
[0457] The stability of Tn3.sup.SS4 to proteolytic degradation was
tested by incubation with thermolysin as previously described in
Example 1 for the wild type protein. In contrast to the wild type
protein, Tn3.sup.SS4 resisted proteolysis, even after overnight
incubation at room temperature (FIG. 10G).
[0458] Thermal unfolding of Tn3.sup.SS4 was assessed by
differential scanning calorimetry (DSC) as previously described. A
1 mg/mL Tn3.sup.SS4 sample in 20 mM sodium phosphate at pH 7.0
exhibited a melting temperature (Tm) of 71.degree. C., which is
26.degree. C. higher than the Tm of the wild type protein under the
same conditions (FIG. 10H). The Tm was slightly elevated for
Tn3.sup.SS4 in 20 mM sodium acetate pH 5.0. At this pH, the Tm for
Tn3.sup.SS4 was 74.degree. C. which is 18.degree. C. higher than
the wild type protein. As with the wild type protein, thermal
unfolding of Tn3.sup.SS4 appeared completely reversible at pH 7.0,
but resulted in precipitation at pH 5.0.
[0459] Size exclusion chromatography with multi-angle light
scattering (SEC-MALS) was used to determine whether Tn3.sup.SS4 was
monomeric in solution, using the same conditions previously
described for the wild type protein. SEC-MALS analysis of a 2.0
mg/mL Tn3.sup.SS4 sample revealed that the protein was completely
in the monomeric state (FIG. 10I). The experimentally-derived
monomer mass was 10.7 kDa which is in close agreement with the
calculated mass of 10.8 kDa.
Preparation of a Dual-Disulfide Containing Tn3 Variant
[0460] Given the enhanced stability of both the Tn3.sup.SS3 and
Tn3.sup.SS4 variants relative to the wild type protein, a new Tn3
variant was prepared to determine whether the stabilizing effect of
each disulfide bond would be additive in the context of a
combination mutant.
[0461] A construct for recombinant expression of this
tetra-cysteine variant, designated Tn3.sup.SS3+4 was prepared by
methods previously described and contained 4 cysteine residues at
positions corresponding to disulfides 3 and 4 (see FIG. 10B).
Protein was expressed and purified as previously described in
Example 1. The yield of purified protein obtained for this variant
was 22 mg from 200 mL E. coli culture. As this is the maximum
binding capacity of the column used for purification, the actual
expression level is at least 110 mg/L.
[0462] Refolding of Tn3.sup.SS3+4
[0463] Purified Tn3.sup.SS3+4 was analyzed by reverse phase HPLC,
and compared to material that was first preincubated with the
strong reducing agent DTT in the presence of guanidine
hydrochloride. While the DTT-treated material chromatographed as a
single peak, untreated Tn3.sup.SS3+4 showed the presence of 3
additional earlier eluting peaks most likely due to partial
formation of either or both potential disulfides bonds (FIG.
10J).
[0464] In order to refold Tn3.sup.SS3+4 to the correct dual
disulfide-containing protein, the sample was diluted to 1 mg/mL
with 6M guanidine hydrochloride containing 10 mM DTT and buffered
at pH 8. After 10 minutes incubation at room temperature, the
sample was dialyzed overnight against 0.5M guanidine hydrochloride
buffered at pH 8.5 with 20 mM Tris.HCl, and containing 4 mM reduced
glutathione. HPLC-analysis of the refolded material indicated
>95% conversion of the reduced protein to a single peak
corresponding to the earliest eluting peak in the unfolded
preparation. This product, by virtue of its elution time profile,
was presumed to contain the 2 correctly formed disulfide bonds.
(FIG. 10J).
[0465] Characterization of Tn3.sup.SS3+4
[0466] Unfolding of Tn3.sup.SS3+4 by guanidine hydrochloride
(GuHCl) at pH 5.0 was determined by fluorescence and compared to
previous data for Tn3 and Tn3.sup.SS4. The Cm for unfolding of
Tn3.sup.SS3+4 at pH 5.0 was between 5.0-5.5M GuHCl (FIG. 10K).
which is considerably higher than the Cm for wild type or
Tn3.sup.SS4. The significant enhancement in stability to
GuHCl-induced denaturation suggests that the stabilizing effects of
disulfides 3 and 4 are additive when both sets of mutations are
combined into the one scaffold.
8.5 Example 5
Identification, Expression and Characterization of Tn3 Structural
Motifs Derived from Hyperthermophilic Organisms
[0467] Tn3 Structural Motif Identification
[0468] A BLAST search was performed to identity putative Tn3
structural motif sequences encoded in the 48 archaeal genomes
within the NCBI database. The search was further restricted to the
genomes of hyperthermophilic organisms, which we define here as
organisms which grow optimally at temperatures of 70.degree. C. or
higher. Tn3 was used as a query sequence, and this led to the
identification of a Tn3 structural motif within a hypothetical
protein from the organism Archaeoglobus fulgidus. This Tn3
structural motif was in turn used as the query sequence to identify
further Tn3 structural motifs, and those in turn were used as query
sequences. A total of 14 potential Tn3-coding sequences were
identified within hypothetical proteins from 5 hyperthermophilic
organisms. The sequences obtained are represented in Section 6
herein.
[0469] Hyperthermophile Tn3 Expression and Purification
[0470] Five of the predicted hyperthermophile-derived Tn3 proteins
were selected for expression in E. coli. Synthetic cDNAs encoding
each of these proteins, and optimized for codon usage in E. coli,
were supplied by GenScript Corporation as per the sequences shown.
Each cDNA contained flanking Nco I and Kpn I restriction sites, and
following digestion with these enzymes, the inserts were cloned
into a modified pET22b vector (Novagen) containing corresponding
Nco I/Kpn I sites. Recombinant expression of the encoded C-terminal
hexahistidine-tagged proteins and purification from E. coli lysates
was performed according to the procedure previously described for
human-derived Tn3 structural motifs. The Tn3 structural motif from
Staphylothermus marinus, and both Tn3 structural motifs from
Sulfolobus tokodaii expressed well, while Tn3 structural motifs
from Sulfolobus solfataricus and Archeoglobus fulgidus expressed at
a low level (FIG. 11A).
[0471] Characterization
[0472] The stability of each Tn3 structural motif, with the
exception of the Tn3 from Sulfolobus solfataricus, was analyzed by
DSC, fluorescence, and thermolysin-treatment as previously
described (See Example 1).
[0473] The thermograms for Tn3 structural motifs at from S.
tokodaii at pH 7.0 did not exhibit a defined peak corresponding to
thermal unfolding. Rather, the data showed that S. tokodaii 1Tn3
precipitated at temperatures above 70.degree. C., while S. tokodaii
1Tn3 precipitated above 50.degree. C. (FIG. 11C). The thermogram
for the Tn3 structural motif from A. fulgidis showed a
characteristic peak with Tm for unfolding of 77.degree. C., as with
the Tn3 structural motif from S. marinus which had a Tm of
83.degree. C. (FIG. 11B). Thermal unfolding was not reversible for
either of these two proteins, which precipitated at temperatures
above Tm.
[0474] Unfolding of the Tn3 structural motifs at pH 7.0 by
guanidine hydrochloride (GuHCl) was analyzed by fluorescence. All
Tn3 structural motifs required high concentrations of GuHCl to
effect unfolding, with the midpoints of unfolding ranging from 4.5M
to 6M GuHCl, as exemplified for the Tn3 from S. marinus (FIG.
11D).
[0475] Stability to proteolysis by thermolysin was analysed using
the same conditions previously described (See Example 1), where
samples of 45 .mu.M Tn3 were incubated with thermolysin at 0.45
.mu.M. All Tn3 structural motifs were resistant to proteolysis,
although rapid cleavage of small 1-2 kDa fragments was observed for
Tn3 proteins from A. fulgidis, S. marinus and S. tokodaii 2Tn3
which we assume are N- and/or C-terminal fragments that do not form
part of the core Tn3 structural motif. A significant proportion of
all 4 core Tn3 structural motifs remained undigested after 16 hr
thermolysin treatment, as exemplified for the Tn3 from S. marinus
and S. tokodaii (2Tn3) (FIG. 11F+G).
[0476] The stability of the Tn3 structural motifs from S. tokodaii
was also assessed at pH 3.0 in 20 mM sodium citrate buffer, given
this organism is acidophilic in addition to being
hyperthermophilic. Both S. tokodaii 1 Tn3 and 2Tn3 were more stable
to GuHCl-induced unfolding at pH 3.0 compared to pH 7.0, with 1Tn3
being more stable than 2Tn3. Thermal unfolding of S. tokodaii 1Tn3
at pH 3.0 in 20 mM sodium citrate was also assessed by DSC.
[0477] In contrast to thermal unfolding at pH 7.0, the thermogram
at pH 3.0 showed a characteristic peak indicating a Tm of
98.degree. C., moreover, unfolding at this pH was partially
reversible. A comparison of thermal and GuHCl-mediated unfolding
for S. tokodaii 1Tn3 is shown in (FIGS. 11C and 11E.)
[0478] Exploiting Stability for Purification of Tn3 Structural
Motifs
[0479] The stability of hyperthermophile-derived Tn3 structural
motifs to extremes of temperature, pH and proteolysis were
exploited to purify these proteins from crude E. coli lysates. The
Tn3 proteins from S. marinus and S. tokodaii (1Tn3) were expressed
in E. coli and soluble lysates prepared as previously described.
Lysate containing S. marinus Tn3 was heated at 70.degree. C. for 15
minutes, precipated protein was removed by centrifugation, then
supernatant was analyzed by SDS-PAGE and compared to lysate which
had not been heated (FIG. 11H). As seen on the gel, a majority of
E. coli contaminants were removed by the heat treatment resulting
in significant purification of the Tn3 protein. Similarly,
treatment of soluble E. coli lysate with thermolysin at 55.degree.
C. for 45 minutes also resulted in significant removal of E.
coli-derived proteins and concomitant purification of the S.
marinus Tn3 (FIG. 11I).
[0480] The pH and high temperature stability of S. tokodaii 1Tn3
protein were utilized to remove E. coli proteins from crude lysate.
Dilution of lysate, which was buffered at pH 7, with 4 volumes of
200 mM sodium citrate pH 3.0 resulted in significant precipitation
of E. coli-derived proteins. After removal of precipitate by
centrifugation, the supernatant was then heated at 70.degree. C.
for 15 minutes, and newly precipitated protein was again removed by
centrifugation. SDS-PAGE analysis of untreated, pH 3-treated, and
pH 3/heat-treated samples shows the dramatic removal of background
E. coli proteins by these 2 steps (FIG. 11J).
8.6 Example 6
Loop Swapping Analysis of SynBP01
Summary
[0481] Three different variants of SynBP01 were constructed to test
if one loop or both the BC and FG loops were contributing to the
binding interface of the Tn3 (FIG. 12). In addition, the
Tn3.sup.SS4 mutation was added to test its affect on binding to
SYNAGIS.RTM.. These variants differed in amino acid sequence from
SynBP01 as follows:
"SynBP01-BC only"--FG loop sequence replaced with RRGDMSSNPA
"SynBP01-FG only"--BC loop sequence replaced with FKPLAEIDG
"SynBP-1 SS4"--substitution of Ile and Ser, shown by line 4 in FIG.
10B, with Cys
[0482] Experimental Procedure
[0483] Phage display vector encoding gene 3 fragment fusions of
SynBP01 and its 3 variants were transformed into E. coli, and these
bacteria were then used to prepare phage displaying each of these
Tn3 proteins as described in Example 3. Plates were coated with
SYNAGIS.RTM. at 10 .mu.g/ml in PBS pH 7.2 overnight at 4.degree. C.
Plates were blocked with PBS containing 0.1% v/v Tween-20 plus 4%
w/v skim milk powder (PBST 4% milk). Diluted phage stocks were
added to column 1 and a 3-fold serial dilution was performed across
the plate using PBST 1% milk as diluent. Plates were incubated at
room temperature for 2 hours with gentle shaking. After washing,
bound phage were labeled with anti-M13 HRP conjugated antibody (GE
Healthcare, Piscataway, N.J.) and detected colorimetrically by
addition of TMB substrate (KPL Laboratories, Gaithersburg, Md.).
The absorbance was read at 450 nm after quenching the color
development by addition of 2.5M phosphoric acid.
[0484] Results: The data presented in FIG. 12 demonstrates that
both the BC and FG loops are required for binding of the Tn3
scaffold to SYNAGIS.RTM.. SynBP01 variants containing a
substitution of either loop with the wild type Tn3 sequence did not
exhibit binding to plate bound SYNAGIS.RTM.. These results
demonstrate that at least two loops of the Tn3 scaffold act
cooperatively to present a functional binding surface.
[0485] In addition, the introduction of the SS4 disulfide bond into
the SynBP01 scaffold ablates binding to SYNAGIS.RTM. (FIG. 12). A
loss of activity for the SS4-containing variant of SynBP01 is
probably due to a subtle conformational effect given this disulfide
is not expected to result in a large change in the structure of a
Tn3 scaffold protein. This further suggests that binding of
SYNAGIS.RTM. is exquisitely dependent on the three dimensional
structure of SynBP01.
8.7 Example 7
Construction of a 2 Loop Library on the Tn3.sup.SS4 Scaffold
[0486] A new library was constructed based upon the Tn3.sup.SS4
scaffold shown in FIG. 10B. The BC loop diversity was introduced
using PCR and the FG loop diversity was introduced using Kunkel
mutagenesis (Table 5). A library of approximately
1.12.times.10.sup.10 members was constructed.
TABLE-US-00030 TABLE 5 Degenerate oligonucleotides for Tn3.sup.SS4
library construction Oligo Loop Sequence Seq ID BC9 BC
ACCGCGCTGATTACCTGGTCTNNKSCGNNKG 217
STNNKNNKNNKGGCTGTGAACTGACCTATGGC BC11 BC
ACCGCGCTGATTACCTGGTCTCCGBSTNNKN 218
NKNNKNNKNNKNNKACCGGCTGTGAACTGACC TATGGC BC12 BC
ACCGCGCTGATTACCTGGGCGVMACCGNNKNN 219 KNNKRRCRGCNNKATTNNKGGTTGTGAACT
GACCTATGGC FG9 FG TATGAAGTGAGCCTGATTTGCNNKAMSNNKN 220
NKGGTNNKNNKNNKAGCAAAGAAACCTTTAC CACC FG10 FG
TATGAAGTGAGCCTGATTTGCNNKAMSNNKN 221 NKNNKNNKRGCAACCCGGCGAAAGAAACCTT
TACCACC FG11 FG TATGAAGTGAGCCTGATTTGCNNKAMSNNKN 222
NKGGTNNKNNKAGCAACCCGGCGAAAGAAACC TTTACCACC
[0487] Experimental Procedure
[0488] The BC loop diversity was made by using the BC9, 11, or 12
primers in Table 5. These primers annealed on their 3' ends to the
Tn3 DNA and the degeneracy formed a library upon completion of the
PCR. These PCR products were amplified with flanking primers to
make a complete Tn3 gene which was then digested with NcoI and KpnI
and ligated into the phage display vector. The DNA was transformed
into E. coli by electroporation. The final diversity of the BC
library was estimated to be about 3.4.times.10.sup.9 members.
[0489] After electroporation, the BC library was incubated for 1
hour at 37.degree. C. with shaking. M13KO7 helper phage was added
and after one hour the cells were diluted to a larger volume and
grown at 37.degree. C. with shaking overnight. The next day phage
were removed and concentrated from the supernatant by precipitation
with PEG 8000.
[0490] BC library phage was used to infect CJ236 E. coli. After a
one hour infection, cells were diluted into 2xYT with 100 .mu.g/mL
carbenicillum and grown overnight with shaking at 37.degree. C. The
next day phage were removed and concentrated from the supernatant
by precipitation with PEG 8000. Single stranded DNA was recovered
by using a Qiagen (Valencia, Calif.) QIAprep spin M13 kit. This DNA
served as the template for Kunkel mutagenesis using the FG primers
in Table 5.
8.8 Example 8
Panning the Two Loop Tn3.sup.SS4 Library for SYNAGIS.RTM. Specific
Scaffolds
[0491] SYNAGIS.RTM. was biotinylated with 15 molar equivalents of
EZ Link sulfo-NHS-SS-biotin (Pierce, Rockford, Ill.). After
incubation for 1 hour at room temperature, the sample was dialyzed
in PBS overnight to remove unconjugated biotin. The next day M280
streptavidin beads (Dynal, Carlsbad, Calif.) and the two loop
library were blocked in PBS containing 10 mg/ml BSA for 1 hour. 10
ug of biotinylated SYNAGIS.RTM. were added to the blocked phage and
incubated at room temperature on an end-over-end rotating mixer for
two hours. SYNAGIS.RTM. was captured with the blocked streptavidin
beads for 30 min on the rotating mixer at room temperature. After
three washes with PBST to remove unbound phage, the bound phage
were eluted with 75 mM DTT. XL-1 Blue E. coli were infected with
eluted phage, co-infected with M13KO7 helper phage and repropagated
overnight as described in Example 3. The next day, phage were
harvested from overnight culture media as described in Example
3.
[0492] The second round of panning was the same as the first with
casein used at 10 mg/ml as the blocking reagent. The beads used for
the second round were Spherotech (Lake Forest, Ill.) avidin-coated
magnetic particles. For the third round, casein was used as the
blocking reagent and M280 streptavidin beads were used to capture
SYNAGIS.RTM.. For the fourth round BSA was used as the blocking
reagent and Spherotech avidin magnetic particles were used to
capture SYNAGIS.RTM.. After 4 rounds of panning, E. coli were
infected with eluted phage and plated. Individual colonies were
cultured in 96 well format, infected with M13KO7, and culture
supernatant was used in a phage ELISA to identify SYNAGIS.RTM.
binding clones. Three new SS4 stabilized clones were identified,
the sequences of which are shown in Table 6.
TABLE-US-00031 TABLE 6 BC and FG loop sequences of SYNAGIS .RTM.
specific binding scaffolds Seq ID Clone BC loop Seq ID NO: FG loop
NO: SYNAGIS .RTM. specific binding scaffolds 4 SPGERIWMFTG 105
PNYERISNPA 106 5 SPSGRVILWTG 107 DNLYGRISNPA 108 6 ATPGCRNGKIVG 109
TTSVGATSNPA 110
8.9 Example 9
Panning the Two Loop Tn3.sup.SS4 Library for TRAIL-R2 Specific
Scaffolds
[0493] M280 streptavidin beads were washed with PBST and
biotinylated goat anti-human IgG Fc fragment specific antibody
(Jackson ImmunoResearch, West Grove, Pa.) was added. After
overnight incubation at 4.degree. C., a control IgG1 antibody or
TRAIL-R2/Fc fusion protein (R & D Systems, Minneapolis, Minn.)
was added, and again incubated overnight at 4.degree. C. The beads
were washed with PBST and blocked in PBST 2% milk prior to use.
[0494] The two loop Tn3.sup.SS4 phage library was incubated
overnight at 4.degree. C. with the control IgG1 antibody-coated
beads to deplete the library of binders to the beads or human IgG1
Fc. The depleted library was then added to TRAIL-R2 coated beads
and incubated for 2 hours at room temperature on a rocking
platform. Beads were washed with PBST and added to XL-1 Blue E.
coli to propagate bound phage as described in Example 8. This
panning procedure was repeated for 4 more rounds, except that
incubation with control antibody beads for background depletion was
performed for 1 hour rather than overnight. Individual clones were
analyzed by phage ELISA after the fourth and fifth rounds of
panning to identify TRAIL-R2 binding variants.
[0495] Following the sequencing of positive clones from the
TRAIL-R2 phage ELISA, nine unique binding clones were identified
(Table 7). A lower case q indicates that a TAG stop codon was at
that position. A suppressor strain such as XL-1 Blue allows for
expression of genes with a TAG stop codon by inserting a glutamine
at this position.
TABLE-US-00032 TABLE 7 BC and FG loop sequences of TRAIL-R2
specific binding scaffolds Clone SEQ ID name BC loop SEQ ID NO. FG
loop NO. 2F4 SPCIMVCLRTG 126 RRGDMSGAPA 127 5B10 SPCLFVCLRTG 128
RRGDMSGAPA 129 10D9 SPPLFCCqKTG 130 FKLTGFLYS 131 6F11 SPSVARMLETG
132 ITLCGRGVS 133 8B3 SPPEYAFYYTG 134 VKNCGLFSNPA 135 5E5 SLAPGYRLG
136 VKLCMRGNPA 137 2H6 ATPSVFDSHIEG 138 WKHHGDAWS 139 7G11
AKPSIVNGFISG 140 DKCFGAMKS 141 6C7 AKPMSCSGYIqG 142 AKLTGWLCS
143
8.10 Example 10
Binding Affinity Determination for a TRAIL-R2 Specific Scaffold
[0496] Goat anti-human-Fc IgG was immobilized at a density of
.about.7700 RUs onto a flow cell of a CM5 Biacore sensor chip
surfaces using a standard amino coupling protocol (BIAcore, Inc.).
Separately, a blank surface was also prepared on the same chip
using the identical coupling protocol, minus the protein. This
blank surface was used as a reference cell throughout the
experiment, and served to correct for both non-specific binding and
certain housekeeping artifacts.
[0497] TRAIL-R2/Fc protein was prepared at 100 nM in instrument
buffer in (HBS-EP buffer, BIAcore, Inc., consisting of the
following: 10 mM HEPES buffer, pH7.4, 150 mM NaCl, 3 mM EDTA, and
0.005% P20.), then injected over both the Fc-capture and control
surfaces at a flow rate of 75 uL/min. Capture levels of the ligand
approximated 800 RUs.
[0498] After baseline stabilization, solutions of the Tn3 clone 5E5
(Table 7) were injected over both the captured ligand and control
surfaces. Between injections, the Fc-capture surface was
regenerated with two 1 min. injections of 10 mM Gly, pH2.
[0499] Several buffer and control protein injections were also
interspersed throughout the injection series. Later, these buffer
injections were used, along with the reference cell data, to
correct the raw data sets for injection artifacts and/or
non-specific `binding` through a technique commonly referred to as
"double-referencing." (Myszka, D. G. (1999) J. Mol. Recognit. 12,
pp. 279-284). Sensorgram overlays of the fully corrected data were
generated using the BIAevaluation 4.1 software (BIAcore, Inc,
Uppsala, Sweden). The affinity of 5E5 for binding to TRAIL-R2 was
calculated by measuring the k.sub.on and k.sub.off.values in the
BIAevaluation software for the sensorgram shown in FIG. 13. This
analysis resulted in anestimated K.sub.d of 700 nM for 5E5 binding
to TRAIL-R2.
8.11 Example 11
Competition for Binding to TRAIL-R2 with Clones 5E5 and 7G11
[0500] Soluble 5E5 and 7G11 (TRAIL-R2 specific clones isolated in
Example 9) were expressed and used in a competition phage ELISA
assay to assess whether they specifically bind TRAIL-R2. TRAIL-R2
coated plates were incubated with phage displaying TRAIL-2 specific
Tn3' sin the presence or absence of soluble Tn3 clones 7G11 or 5E5.
As shown in FIG. 14, soluble 5E5 competes with phage displayed 5E5
and all other phage displayed clones except 2116 and 7G11. Soluble
7G11 only competes with phage displayed 7G11 and 2H6. This
experiment indicates that all clones are specific for TRAIL-R2 and
that there are two different epitopes on TRAIL-R2 recognized by
this panel of Tn3 proteins.
8.12 Example 12
Construction of a Three Loop Tn3.sup.SS4 Library
[0501] A phage displayed three loop library based on the
Tn3.sup.SS4 scaffold was made by randomizing the sequences of the
BC, DE, and FG loops using the primers shown in Table 8. Briefly,
single stranded DNA from the two loop Tn3 BC loop library with
Tn3.sup.SS4 (from Example 7) was used as a template for a PCR with
the DE rev primer in Table 8. This PCR generated a product that
contained a portion of the Tn3 gene with BC and DE randomization. A
second PCR used this BC, DE loop randomized PCR product as template
for amplification with the FG primers listed in Table 8. The
resulting PCR products were amplified with flanking primers to make
a complete Tn3 gene which was then cut NcoI to KpnI and ligated
into the phage display vector. The DNA was transformed into E. coli
by electroporation.
[0502] After electroporation, the library was incubated for 1 hour
at 37.degree. C. with shaking. M13KO7 helper phage was added and
after one hour the cells were diluted to a larger volume and grown
at 37.degree. C. with shaking overnight. The next day phage were
purified from the culture supernatant by precipitation with a
saline PEG 8000 solution. The library size was estimated to contain
about 1.5.times.10.sup.9 members based on the number of E. coli
transformants.
TABLE-US-00033 TABLE 8 Degenerate oligonucleotides for three loop
Tn3.sup.SS4 library construction SEQ Sequence Name Sequence ID: DE
rev CCGGTTTCAGGTTACCAATGCTATAMNNMNNMN 223
NMNNMNNMNNCAGATCTATGGTGGTGCGATCGCC FG9 rev
CCGCCACCGGTGGTAAAGGTTTCTTTGCTMNNM 224
NNMNNACCMNNMNNSKTMNNGCAAATCAGGCTC ACTTCATATTCGG FG10 rev
CCGCCACCGGTGGTAAAGGTTTCTTTCGCCGGG 225
TTGCYMNNMNNMNNMNNSKTMNNGCAAATCAG GCTCACTTCATATTCGG FG11 rev
CCGCCACCGGTGGTAAAGGTTTCTTTCGCCGGGT 226
TGCTMNNAMNNACCMNNMNNSKTMNNGCAATCA GGCTCACTTCATATTCGG
8.13 Example 13
Panning the Three Loop Tn3.sup.SS4 Library for TRAIL-R2Specific
Scaffolds
[0503] The three loop Tn3.sup.SS4 library constructed in Example 12
was panned against TRAIL-R2, and specific clones identified in by
phage ELISA as described in Example 9 In total, 19 new Tn3s were
identified that bound to TRAIL-R2 (Table 9).
TABLE-US-00034 TABLE 9 BC, DE, and FG loop sequences of TRAIL-R2
specific binding scaffolds Seq Seq Seq Clone BC loop Id. DE loop
Id. FG loop Id. 1E03 AAPFFGSSYISG 144 HYYVTR 145 VNLSGHMPS 146 2B04
APPMLTDSEING 147 TSSYWS 148 STLRRNAIS 149 1C12 AKPEKWDGSIYG 150
NSRHTA 151 FTPYGAKSNPA 152 1A03 APPPFSNSCIIG 153 RPGRAS 154
STGTGLPSNPA 155 1C10 SPCCPYDRYTG 156 QSSRSH 157 ITTFGHVSNPA 158
1B12 AKPRqGGSNISG 159 YHKGLH 160 PKMTGYTYS 161 2G03 SPGPLLRHTTG 162
RPIPRA 163 RNRPQqSNPA 164 2D3 SPGGFqKITTG 165 VNRRNH 166 LTYKARAIS
167 1C06 SPRMYTWIqTG 168 THLSGS 169 LKLTRTHIS 170 2F08 SHAGGIRIG
171 HVVVqVY 172 MTPYLLGNPA 173 1B04 SPSHGVESSTG 174 HGLqRV 175
AKICGHLVS 176 3B11 SPCqLLALITG 177 NSRHYH 178 YTSTGQRSNPA 179 1D8
SPCqMLSSLTG 180 NIERPK 181 FTMTGYRSNPA 182 2A12 SPCCqEFTLTG 183
HNHHHH 184 ITDAGNKSNPA 185 1E05 SPCSPCqLVTG 186 SCTRAK 187
1IKLGDTSNPA 188 2F02 SPSRGGTSLTG 189 DqVRAT 190 HTNSGqPSNPA 191
1H05 SPGMFDqVRTG 192 GKYWER 193 RNQYGqHqS 194 2A11 SPPFRAGHVTG 195
VTARCq 196 TTGNGLRSNPA 197 1G11 SWAqANPGG 198 WHSITF 199 KTKVqSSNPA
200
[0504] The amber stop codon in the nucleotide sequences of clones
2D3 and 1 G11 was replaced with a glutamine codon by site-directed
mutagenesis. These Tn3 clones, along with 1E3, 1C12, and 2B4 were
cloned into a E. coli expression vector (described in Example 1)
and transformed into BL21 DE3 cells. After induction with IPTG, the
transformed bacteria were grown for 5 hours at 30.degree. C. The
cells were pelleted, lysed by sonication, and the soluble fraction
was purified on a HiTrap chelating HP column (GE Healthcare,
Piscataway, N.J.). Tn3 clones 2B4 and 1C12 were obtained in good
yield, however, a poor recovery of the remaining three clones was
the result of overexpression leading to accumulation of the
proteins into inclusion bodies. In this case, a high yield of Tn3
was subsequently obtained by solubilizing the inclusion bodies in
buffered 6M guanidine hydrochloride (GuHCl), then purifying on a
HiTrap chelating column under denaturing conditions. All Tn3s were
subsequently refolded by dialysis of reduced and denatured samples
into native buffer in the presence of a cysteine/cystine redox
pair.
8.14 Example 14
Determining Epitope Diversity
[0505] A selection of the phage displayed clones from Table 9 were
tested to see if soluble forms of Tn3s 1E3, 1G11, 2B4, 1C12 and 2D3
would compete for binding to TRAIL-R2 in a phage ELISA. The assay
was performed in a similar manner to the competition for binding in
Example 11, and the results are shown in FIG. 15 A, B, and C.
Soluble Tn3 clones 1E3, 1C12, and 2D3 significantly inhibited the
binding of most of the phage linked Tn3s to TRAIL R2, with the
exception of clones 8B3 and 7G11. Soluble 1G11 did not
significantly compete with any of the phage bound clones. Soluble
2B4 showed little to moderate inhibition in most cases. The fact
that 1E3, 1C12, and 2D3 competed with the same set of phage linked
Tn3s indicates that these three soluble Tn3s and the phage linked
Tn3s likely bind to the same epitope on TRAIL-R2.
8.15 Example 15
Cell Viability Assays Using Tn3 Monomers Linked to an Anti-His
Monoclonal Antibody
[0506] Colo 205 is a cell line which is highly sensitive to
TRAIL-induced killing. TRAIL-R2 binding Tn3s, when multimerized via
binding to a complex of mouse anti-His tag antibody and anti-mouse
IgG, were tested for their ability to induce killing of Colo205
cells. Colo205 cells in 100 ul of RPMI 1640 medium with 10% FBS
were plated into each well of a flat bottom 96-well culture plate
and incubated overnight at 37.degree. C. Tn3 proteins 7G11 and 5E5
(Table 7), 1C12, 2D3 and 1E3 (Table 9) and Tn3.sup.SS4 (Example 4)
were incubated with mouse anti-His tag antibody (Penta-His; Qiagen
Inc) and rabbit anti mouse IgG in a molar ratio of 2:1:0.5. Serial
dilutions of each Tn3 complex was made in RPMI 1640 medium
containing 10% FBS to a final concentration of 5 .mu.M, 1.66 .mu.M,
0.55 .mu.M, 0.185 .mu.M and 0 .mu.M based on the Tn3 content. After
removal of medium from cells cultured overnight, 100 of the
Tn3-antibody complexes was added. Each assay point was performed in
triplicate.
[0507] After addition of Tn3 complexes, the cells were incubated
for 3 days in at 37.degree. C., after which cell viability was
measured in a CellTiter-Glo.RTM. luminescent cell viability assay
(Promega Corp., Madison, Wis.) according to the manufacturers
instructions. The percent viability for cells treated with a
TRAIL-R2 binding Tn3s was calculated by dividing the luminescent
signal obtained in the CellTiter-Glo.RTM. assay by the
corresponding signal obtained for cells treated with the same
concentration of non TRAIL-R2 binding control Tn.
[0508] The cell viability assay (FIG. 16) showed that Tn3 clones
5E5, 1C12 and 2D3, when multimerized via antibody complexation,
were able to inhibit Colo205 viability in a dose-dependent manner
relative to treatment of cells with a non-TRAIL-R2 binding Tn3.
Inhibition of cell viability was presumably due to cellular
apoptosis triggered by TRAIL-R2 ligation, although pathway-specific
assays would be needed to confirm this. Clones 7G11 and 1E3 did not
show detectable activity at the concentrations used in the assay.
Additional cell assays showed that none of the active Tn3 proteins
affected cell viability when assayed without an anti-His tag
capture antibody. This demonstrates that activity is dependent on
the presentation of multimerized TRAIL-R2 binding moieties. Clone
5E5 also lacked activity if the anti-IgG antibody was not present,
however, clones 1C12 and 2D3 were active in the absence of
anti-IgG, suggesting that dimeric presentation via anti His-tag
antibody capture is sufficient to trigger TRAIL-R2 signaling.
8.16 Example 16
Construction of Polyvalent Anti TRAIL R2Tn3 Antibody Fusions
[0509] Given the requirement for presentation of oligomeric Tn3
complexes to effect TRAIL-R2-dependent cell killing, Tn3 fusion
constructs were designed for the production of bi- and tetravalent
Tn3-containing proteins (FIG. 17). A bivalent Tn3 construct was
designed by fusion of Tn3 to the Fc region of human IgG1, while a
two chain tetravalent Tn3 construct was designed based on
co-expression of a Tn3-C.kappa. fusion with Tn3-IGHG1, i.e Tn3
fused to a human C-kappa region, and to the heavy chain constant
region of human IgG1. The latter construct is similar in nature to
an antibody, except that the light and heavy chain variable regions
were replaced with a Tn3 moiety.
[0510] The constructs shown in FIGS. 17A and B, were expressed in
293F cells transiently transfected with the in-house pOE expression
vector coding for each of the proteins. After 10 days in culture,
media (250 mL) was harvested, and the protein was purified by
protein A affinity chromatography. Presented in Table 10 are the
expression levels of a selection of multivalent Tn3 constructs.
1C12 and a control SYNAGIS.RTM. binding Tn3 named D1 (from Example
8 Seq ID NO: 105 and Seq ID NO 106) expressed well as either the Fc
fusion, or two chain Tn3-C.kappa./Tn3-IGHG1 fusions. Hybrid
tetravalent fusions of 1C12 and 2D3 (1C12 linked to IGHG1, 2D3
linked to C.kappa. and vice versa) along with the 2D3 Fc fusion
yielded lower amounts of material, while the two chain
2D3-C.kappa./2D3-IGHG1 fusion did not express SDS-PAGE analysis of
the protein A purified proteins is shown in FIG. 18.
TABLE-US-00035 TABLE 10 Yield of Different Fc fusion and
Tetravalent Antibody Fusion Constructs # Name Final 1 D1-Fc 15 mg 2
1C12-Fc 15 mg 3 2D3-Fc 3.5 mg 4 D1-C.kappa./D1-IGHG1 13 mg 5
1C12-C.kappa./1C12-IGHG1 10 mg 6 2D3-C.kappa./1C12-IGHG1 4 mg 7
1C12-C.kappa./2D3-IGHG1 4 mg
[0511] Biosensor Assays of 1C12, 1C12 Fc, and 1C12 Tetravalent
[0512] A qualitative comparison of binding by monomeric 1C12,
1C12-Fc and 1C12-C.kappa./1C12-IGHG1 proteins was performed by
injecting samples of each of these proteins over a TRAIL-R2 chip on
an Attana biosensor instrument. As shown in FIG. 19, complexes of
TRAIL-R2 with oligomerized forms of 1C12 show a substantial
improvement in affinity relative to monomeric 1C12. The specificity
of binding was demonstrated by injection of D1-Fc and
D1-C.kappa./D1-IGHG1proteins which did not interact with
immobilized TRAIL-R2. The dissociation rates for the different 1C12
constructs followed the order
1C12>1C12-Fc>1C12-C.kappa./1C12-IGHG1, consistent with the
bi- and tetravalent constructs exhibiting avidity in their
interaction with TrailR2.
8.17 Example 17
Cell Viability Assays Using Polyvalent Anti TRAIL-R2Tn3 Fusion
Proteins
[0513] H2122 cells were plated in 96 well plates at a density of
10000 cells/well in 50 ul of complete medium (RPMI 1640 medium
supplemented with 10% FBS). Cells were incubated overnight at
37.degree. C. The next day, polyvalent Tn3 fusion proteins (Fc
fusions or two chain tetravalent constructs) alone or in
combination with goat anti-human Fc were serially diluted in
complete medium. To achieve a dose curve, a 3-fold dilution scheme
was used (highest final concentration was 3.6 uM). The goat
anti-human Fc was added at a 1:2 molar ratio (i.e. half of the
concentration of the Tn3-containing molecule). Tn3 and anti-human
Fc alone and in combination were prepared at a 2.times.
concentration (50 ul of each treatment were added per well). All
treatments were done in triplicate wells. Commercially available
TRAIL ligand (Chemicon Cat# GF092) was used as a positive control
for Trail-induced cell death. The final concentrations for Trail
were 1, 0.1, and 0.01 nM. After 48 hrs, the CellTiter-Glo kit from
Promega was used to determine cell viability. Briefly, cells are
allowed to equilibrate for about 10 min to room temperature.
CellTiter-Glo buffer and substrate were mixed to prepare the
CellTiter-Glo reagent as indicated by manufacturer. Each well
received 100 ul of the CellTiter-Glo reagent and the plate was
incubated for 10 min at room temperature prior to reading
luminescence in a Wallac Plate reader. Results are shown in FIGS.
20-22. Each of the 1C12 and 2D3 containing polyvalent constructs
were able to inhibit the viability of H2122 cells, presumably by
activating TRAIL-R2 dependent apoptosis, moreover this activity was
not dependent on higher order crosslinking via coordination with an
anti-Fe antibody. The tetrameric form of 1C12 was more potent in
the cell assay than its dimer form (compare FIGS. 20 and 21),
consistent with the killing activity being a function of the
valency. Fc-cross-linking did not increase the potency of killing
and appeared to reduce the activity of the monospecific 1C12
constructs. Neither D1-Fc nor D1-C.kappa./D1-IGHG1 control
proteins, which do not bind TRAIL-R2, affected cell viability. The
bispecific tetravalent constructs (FIG. 22) had the greatest
potency in inhibiting H2122 cell viability, and this increased
slightly if co-incubated with anti-Fc antibody. The improvement in
activity for the 2D3/1C12 bispecific constructs relative to
monospecific 1C12-C.kappa./C12-IGHG1 may be due to superior potency
for 2D3 vs 1C12 Tn3 units, or because 2D3 and 1C12 recognize
different epitopes on TRAIL-R2 which could result in higher order
aggregation of cell surface TRAIL-R2.
8.18 Example 18
Bacterial Secretion of Tn3 Scaffolds
[0514] A bacterial expression vector was designed to secrete
correctly folded Tn3 scaffold in E. coli. This system would allow
for correct disulfide bond formation within Tn3 and therefore avoid
the refolding process that is required for material expressed
intracellularly as described in Example 4. To create a secretion
vector, an intracellular Tn3 expression vector, similar to that
described in Example 4, but containing a Ptac promoter instead of
T7, was modified by insertion of the signal peptide sequence from
E. coli oligopeptide binding protein (oppA). This signal sequence,
cloned immediately upstream of Tn3, was chosen because oppA is a
highly expressed E. coli protein. An extended 8.times.His tag was
encoded downstream of Tn3 to facilitate purification. To simplify
the transfer of Tn3 cassettes between this and other plasmids, a
modified form of this vector was also created by introducing an Nco
I site at the 3' end of the oppA signal sequence. This modification
results in a single amino acid substitution (L25M) at the
penultimate position within the oppA signal sequence (FIG. 23).
These vectors were referred to as pSec-oppA-Tn3 and
pSec-oppA(L25M)-Tn3.
[0515] Superbroth media containing carbenicillum (100 ug/mL, 1%
glucose) was innoculated with E. coli BL21 DE3 cultures transformed
with pSec-oppA-Tn3 or pSec-oppA(L25M)-Tn3. Cultures were grown at
37.degree. C. to an OD of 0.5-0.8 then induced with 0.2 mM IPTG.
After shaking at 37.degree. C. for 5 hours, cells were separated
from the media by centrifugation. Periplasmic extracts were
prepared by resuspending the cell pellet in 1/10 volume of ice-cold
extraction buffer (10 mM Tris, pH 8 and 1 mM EDTA), incubating on
ice for 10 min, then centrifuging to remove cells. Samples of
periplasmic extract and media were analyzed by SDS-PAGE. Tn3 could
be detected in both media and periplasmic fractions, and expression
levels were similar for constructs containing the wild type or L25M
oppA signal peptides (FIG. 24A). As pSec-oppA(L25M)-Tn3 contains a
convenient 5' Tn3 cloning site, this construct is preferred for the
expression of Tn3 clones derived from display libraries.
[0516] Purification of Tn3 from media was effected by precipitating
the secreted protein with 65% w/v ammonium sulfate, resuspending
the pellet in 50 mM Tris pH 8 buffer, then purifying on a HiTrap
chelating column charged with Ni.sup.2+as previously described.
SDS-PAGE analysis of the purified sample is shown in FIG. 24B.
Purified Tn3 was analyzed by reverse phase HPLC (as described
herein), either with or without DTT pretreatment to reduce any
disulfide bonds. Tn3 eluted as a single peak, and the elution time
shifted after reduction with DTT indicating the purified sample
contained a disulfide bond as expected (FIG. 23C). Non-reducing
SDS-PAGE and size exclusion chromatography of this material were
consistent with a single monomeric species, and mass spectrometric
analysis gave a molecular weight. of 10,896 Da which is within 3 Da
of the predicted molecular weight for the mature,
disulfide-containing sequence shown in FIG. 28A.
[0517] Finally, the expression level of secreted Tn3 in media was
determined in a biosensor assay. Anti-His tag antibody (Penta-His,
Qiagen Inc.) was immobilized onto an Attana A100 carboxyl sensor
chip via standard amine coupling. E. coli BL21 DE3 were transformed
with pSec-oppA(L25M)-Tn3, and protein expression was induced as
described above. Dilutions of clarified media were injected over
the chip, and levels of bound His-tag containing Tn3 were compared
to that generated from injection of purified Tn3 standard. By this
technique, the level of Tn3 detected in crude media was 250
mg/L.
8.19 Example 19
Generation of SynBP01-Fc Fusion
[0518] Summary: A chimeric fusion of a SYNAGIS.RTM.-binding Tn3 and
the Fc region of IgG1 was generated.
[0519] Methods: Expression of SynBP01-Fc
[0520] As described above, SynBP01 is a Tn3 variant which was
identified from a library of BC loop and FG loop-randomized Tn3
variants panned against SYNAGIS.RTM.. Flanking NheI and KasI sites
were introduced by silent mutagenesis and utilized to subclone this
construct into the pOE-Fc vector. The pOE-Fc vector contains the
CH2 and CH3 domains of IgG1 Fc, which is 3' to the KasI restriction
sites. The vector generated by subcloning SynBP01 into pOE-Fc was
named pOE-SynBP01.
[0521] 293EBNA cells (120 mL culture volume) were transfected with
pOE-SynBP01 through Lipofectamine 2000 (Invitrogen), using standard
methods. Supernatant was harvested 10 days post-transfection, and
SynBP01-Fc was purified through Protein A affinity chromatography
(GE Healthcare), eluting with 0.1M glycine, 0.15M NaCl, pH 3.08,
and neutralizing with Tris-HCl buffer at pH 8. The purified sample
was then dialyzed against PBS buffered at pH 7.2. The yield of
purified SynBP01-Fc was 4 mg, indicating an expression level of 33
mg/L.
[0522] BIAcore Analysis of SynBP01-Fc
[0523] BIAcore analysis of this molecule was conducted on a BIAcore
3000 (GE Healthcare), as described in Example 3. This experiment
was designed to determine whether the SynBP01-Fc was competent to
bind SYNAGIS, and to detect qualitatively the difference in
apparent affinity between the original SynBP01 binder and the Fc
fusion.
[0524] Results: When injected at equivalent 1 .mu.M concentrations,
SynBP01-Fc yielded an approximately 6-fold increase in total
response units compared to SynBP01 (FIG. 26). Moreover, SynBP01-Fc
had a substantially reduced off-rate relative to SynBP01, due
primarily to the increased avidity of the bivalent Fc fusion.
Although the dissociation constant (K.sub.D) of the
SynBP01-Fc/SYNAGIS.RTM. interaction was not determined in this
experiment, as the binding surface had not been prepared for a
kinetic analysis, it is evident that the K.sub.D is improved from
the K.sub.D of 16 .mu.M seen with the SynBP01/SYNAGIS.RTM.
interaction in Example 3.
8.20 Example 20
Site-Specific PEGylation of STn3 Scaffold
[0525] Modification of a protein through PEGylation is frequently
used to improve its therapeutic properties, such as decreased
immunogenicity, improved pharmacokinetics and bioavailability by
increasing the effective size of small proteins so as to avoid
renal clearance. Site-specific modification with PEG, that is
attachment at one or more specific residues in the protein, can
avoid inactivation of a target proteins activity that could
otherwise result from attachment of PEG at or near a functional
site within a protein. To demonstrate site-specific PEGylation of a
Tn3-like scaffold protein, a cysteine residue was engineered at the
C-terminus of STn3 (S. tokodaii 1Tn3 from Example 5). As the wild
type sequence of STn3 does not contain cysteine, treatment of the
engineered scaffold with a Cys-specific PEGylation reaction would
lead to site-specific attachment of PEG. STn3 had previously been
expressed as a fusion to a C-terminal 6.times.His tag, in a variant
of the pET-22b vector (Novagen). This vector contains the linker
sequence GGGLE between the protein and the His tag. A variation on
the QuikChange (Stratagene) mutagenesis method was used to mutate
the leucine residue in the linker to a cysteine, which can be
modified by a maleimide reagent. This protein is referred to as
STn3(CTC), and was expressed and purified from BL21(DE3) cells
using an IMAC column as described in Example 1.
[0526] The PEGylation reagent, Sunbright ME-200MA (NOF), was added
to STn3(CTC) at a 4:1 molar excess (PEG reagent:protein), and was
allowed to incubate at room temperature for 72 hours. Protein
PEGylation was monitored by SDS-PAGE (FIG. 27A, lane 5), which
revealed that the majority of the protein had been PEGylated by a
single PEG moiety. As PEGylation typically has the effect of
masking surface charge of a protein, the pH for cation-exchange
chromatography was lowered from 6.0 (for wild-type purification) to
4.5, to ensure that the protein would effectively bind the column.
The protein was purified on a 1 mL SP XL column (GE Healthcare)
using 50 mM acetic acid, pH 4.5. A sodium chloride gradient was
used to elute the protein, with the protein peak occurring at
approximately 120 mM NaCl. Successful removal of residual
unPEGylated STn3(CTC) from the PEGylated product is demonstrated by
SDS-PAGE analysis of fractions obtained from the cation exchange
purification fractions are shown in FIG. 27B, lanes 1-5.)
8.21 Example 21
Analysis of AB, CD and EF loops and Design of Randomized
Library
[0527] To design Tn3 libraries which were randomized in the AB, CD
and EF loops, a bioinformatic analysis was performed to derive
information regarding the length and sequence diversity of these
loops in naturally occurring Fn3 domains. Due to the difficulty in
predicting the AB, CD and EF loop regions based on sequence
information alone, the three dimensional structures of 103
different Fn3 domains from the pdb database were superimposed, and
this was used to align the corresponding amino acid sequences (data
not shown). The locations of the loops regions were used to extract
length and sequence diversity information for each of the loops.
The variation in length for each of the AB, CD and EF loops is
shown graphically in FIG. 28.
[0528] As with loops on the opposite side of the Tn3 molecule, the
AB, CD and EF loops vary in length and sequence composition for
different Fn3 domains. The AB and CD loops are usually 5 to 9 amino
acids long, although exceptions occur for some Fn3 domains which
have AB and/or CD loops longer or shorter than this. The most
common length within this data set was 6 residues for the CD loop
(31% of sequences), and 7 residues for the AB loop (61% of
sequences). Length variation occurs less frequently for the EF
loop, and an 8 residue loop is most commonly observed (80% of
sequences). Both the AB and CD loops show significant diversity in
sequence and do not show overt preferences for specific amino acids
in particular positions. An exception is the final position in the
AB loop which is often Ser or Thr (58/103 sequences). The sequences
of EF loops reveals strongly preferred amino acids at specific
positions, though this is restricted to those that are 8 residues
long. A Leu at position 3 within these loops is strongly conserved
(76/82 sequences), and given the sidechain of this residue is
buried in each of the structures, it is likely to be important for
the structural integrity of the scaffold. A Pro residue is also
commonly observed at position 5 (44/82 sequences), while Gly, Asn,
Asp and Ser are often in position 2 (71/82 sequences) and Thr in
position 7 (40/82).
[0529] A further practical consideration in the design of these Tn3
libraries was to identify an alternative to the "NNK" (N=A, G, T,
C; K=G, T) mixed codon scheme typically used in degenerate
oligonucleotides to code for any amino acid. Although the "NNK"
mixture gives 32 different codons which code for all 20 amino
acids, they are not encoded equally (Table 11). For instance, 3/32
codons in the "NNK" scheme code for Leu (CTG, CTT, TTG), but only
1/32 codes for Asp (GAT). In addition, the "NNK" mixture encodes
one stop codon (TAG) and a Cys codon (TGT), neither of which is
desirable when generating naiive libraries. In considering an
alternative scheme, we took note of the fact that synthetic
antibody libraries have been described which encode CDR sequences
composed of a small subset of amino acids. Antibody libraries with
CDR's composed of just 4 amino acids (Tyr, Ala, Asp, Ser), or even
a binary pair (Tyr, Ser) have been shown to yield specific high
affinity mAbs to protein antigens (Fellouse et al., Proc. Natl.
Acad. Sci. USA. 2004, 101: 12467-72; J. Mol. Biol. 2005, 348:
1153-62). Similarly, a library of scaffold proteins with randomized
loop sequences comprising just Tyr and Ser also yielded specific
binders to a protein target (Koide et al., Proc. Natl. Acad. Sci.
USA. 2007, 104: 6632-7). Although libraries containing highly
restricted sets of amino acids are able to produce specific binding
proteins, it is likely that the diversity of binders that are
obtained from such a library will be limited. We therefore designed
an alternate "NHT" mixed codon scheme for introducing diversity
into a Tn3 library (H=A, T, C). "NHT" mixes code for a reasonable
subset of the 20 amino acids, but avoid the disadvantages described
with "NNK" mixed codons (Table 12). This scheme generates 12 codons
that code for 12/20 amino acids, that is, each codon codes for a
unique amino acid. Moreover, there are no stop or Cys codons.
TABLE-US-00036 TABLE 11 Amino acids encoded by "NNK" codon mixtures
A AAG = Lys ATG = Met ACG = Thr AGG = Arg G AAT = Asn ATT = Ile ACT
= Thr AGT = Ser T G GAG = Glu GTG = Val GCG = Ala GGG = Gly G GAT =
Asp GTT = Val GCT = Ala GGT = Gly T C CAG = Gln CTG = Leu CCG = Pro
CGG = Arg G CAT = His CTT = Leu CCT = Pro CGT = Arg T T TAG = STOP
TTG = Leu TCG = Ser TGG = Trp G TAT = Tyr TTT = Phe TCT = Ser TGT =
Cys T A T C G
TABLE-US-00037 TABLE 12 Amino acids encoded by "NHT" codon mixtures
A AAT = Asn ATT = Ile ACT = Thr G GAT = Asp GTT = Val GCT = Ala C
CAT = His CTT = Leu CCT = Pro T TAT = Tyr TTT = Phe TCT = Ser A T
C
[0530] The final design for Tn3 libraries containing randomized AB,
CD and EF loops is shown below. This design incorporates diversity
observed in natural Fn3 sequences, two different lengths for the AB
and CD loops, and uses "NHT" codon mixes.
[0531] AB loop (7 and 9 residues):
[0532] Tn3 wild type amino acid sequence: KDVTDTT
[0533] Library amino acid sequence (7 aa): Kxxxxxa
[0534] DNA sequence: AAA-NHT-NHT-NHT-NHT-NHT-RST
[0535] Library amino acid sequence (9 aa): Kxxxxxxxa
[0536] DNA sequence:AAA-NHT-NHT-NHT-NHT-NHT-NHT-NHT-RST
[0537] CD loop (7 and 9 residues):
[0538] Tn3 wild type amino acid sequence: KDVPGDR
[0539] Library amino acid sequence (7 aa): xxxxxxx
[0540] DNA sequence: NHT-NHT-NHT-NHT-NHT-NHT-NHT
[0541] Library amino acid sequence (9 aa): xxxxxxxxx
[0542] DNA sequence: NHT-NHT-NHT-NBT-NHT-NHT-NHT-NHT-NHT
[0543] EF loop (8 residues):
[0544] Tn3 wild type amino acid sequence: GNLKPDTE
[0545] Library amino acid sequence: xbLxPxcx
[0546] DNA sequence: NHT-RRB-CTG-NHT-CCG-NHT-RBT-NHT
[0547] Amino acid codes: x=N/D/H/Y/I/V/L/F/T/A/P/S; a=S/T/A/G;
b=N/K/S/R/D/E/G; c=I/T/SN/A/G
[0548] Nucleotide codes: N=G/A/T/C; H=A/T/C; R=A/G; S=G/C;
B=T/C/G
8.22 Design, Expression and Characterization of Charge Engineered
Tn3 Variants with Enhanced Stability Design of Charge Engineered
Tn3 Variants
[0549] The stability of Tn3 to thermal unfolding is greater at pH 5
compared to pH 7, and greater in pH 7 buffer containing 1M salt
than the same buffer without salt (FIG. 29). As high salt
concentrations can mask surface protein charge, while buffer
acidification can result in neutralization of negatively charged
Asp and Glu side chains, these observations suggest that surface
negative charge on Tn3 has a destabilizing effect.
[0550] To explore the potential for enhancing the stability of Tn3
through engineering of surface charge, the locations of Asp and Glu
side chains were mapped onto the three dimensional structure of
Tn3. From a total of 18 Asp and Glu residues contained in Tn3 (SEQ
ID 1), a panel of 8 mutants were designed in which individual Asp
or Glu residues were replaced with the neutral isoteric residues
Asn or Gln (FIG. 30A). The selection of the 8 substitution sites
was biased towards Asp and Glu residues that were in close
proximity to another Asp or Glu, given proximity of like charges
can contribute to destabilization through electrostatic
repulsion.
Charge Mutant Generation and Recombinant expression
[0551] Expression constructs for Tn3 mutants were generated by
site-directed mutagenesis of the wild type expression construct as
described previously. Recombinant protein was expressed in E. coli
and purified by immobilized nickel chelate affinity chromatography
as described previously. All Tn3 mutants expressed at high levels
and were readily purified, although the preparation of the E54Q
mutant did contain some impurities as evidenced by SDS-PAGE
analysis (FIG. 30B).
[0552] Characterization of Stability
[0553] Unfolding of charge mutants by urea was monitored by
intrinsic fluorescence as previously described for wild type Tn3.
To facilitate a comparison of urea-induced unfolding profiles of
wild type and charge mutants of Tn3, the relative fluorescence
emission intensity at 360 nm was plotted as a function of urea
concentration for each protein.
[0554] A comparison of urea-induced unfolding at pH 7.0 for wild
type Tn3 and the various charge mutants (FIG. 31A) showed that 3 of
the mutants (D40N, E54Q and E67Q) had the same or slighty lower
stability than the wild type protein. Five of the mutants (E33Q,
D49N, E52Q, D53N, E86Q) showed small but clearly defined increases
in the midpoints of urea-induced unfolding, suggestive of an
increase in protein stability. The concentration of urea required
to induce 50% unfolding was approximately 0.5M higher for best 3
mutants (E33Q, D49N and E86Q) than for wild type Tn3.
Preparation and Analysis of Tn3 Variants Containing Multiple Charge
Mutations
[0555] Given the enhanced stability of a number of the Tn3 charge
variants, new Tn3 variants containing combined charge mutations
were prepared to investigate whether additive improvements in
stability could be obtained. To this end, three new variants were
prepared containing paired mutations (D49N/E86Q and E33Q/D49N) or a
triple mutation (E33Q/D49N/E86Q). These mutants were recombinantly
expressed and purified, and their urea denaturation profiles were
characterized by fluorescence.
[0556] When compared to the single charge mutants of Tn3, each of
the combination mutants showed further enhancement of stability as
determined by the increase in urea concentration required for
unfolding (FIG. 31B). While the midpoint of urea-induced unfolding
of the individual charge mutants E33Q, D49N and E86Q occurred at
.about.2.5M urea, the midpoint of unfolding for each of the
D49N/E86Q, E33Q/D49N and E33Q/D49N/E86Q mutants corresponded to
.about.3.0M urea. This indicates that combined replacement of
multiple destabilizing Asp or Glu residues can lead to an additive
improvement in Tn3 stability, although in the examples studied
here, a triple mutant was no more stable than a double mutant.
[0557] To further characterize the stabilities of the combination
charge mutants relative to wild type Tn3, these proteins were
analyzed by DSC at pH 7 as previously described. This analysis
further confirmed that the charge mutations led to an improvement
in the stability of the Tn3 scaffold. While the wild type protein
had a mid-point of thermal unfolding (T.sub.m) of 45.degree. C.,
the E33Q/D49N Tn3 mutant had a Tm of 50.degree. C., while D49N/E86Q
and E33Q/D49N/E86Q mutants had Tm's of 52.degree. C. (FIG. 32).
[0558] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above may be used in various
combinations. All publications, patents, patent applications, or
other documents cited in this application are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication, patent, patent application, or
other document were individually indicated to be incorporated by
reference for all purposes.
Sequence CWU 1
1
241190PRTArtificialSynthetic construct 1Arg Leu Asp Ala Pro Ser Gln
Ile Glu Val Lys Asp Val Thr Asp Thr1 5 10 15Thr Ala Leu Ile Thr Trp
Phe Lys Pro Leu Ala Glu Ile Asp Gly Ile 20 25 30Glu Leu Thr Tyr Gly
Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile 35 40 45Asp Leu Thr Glu
Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro 50 55 60Asp Thr Glu
Tyr Glu Val Ser Leu Ile Ser Arg Arg Gly Asp Met Ser65 70 75 80Ser
Asn Pro Ala Lys Glu Thr Phe Thr Thr 85 90294PRTArtificialSynthetic
construct 2Ser Glu Pro Gln Asn Leu Lys Ala Thr Ala Gly Asn Asn Asn
Ile Thr1 5 10 15Leu Thr Trp Asp Pro Pro Ile Asp Asp Gly Gly Cys Arg
Ile Val Glu 20 25 30Tyr Arg Ile Tyr Arg Gly Thr Asn Asn Asn Asn Leu
Glu Tyr Tyr Ala 35 40 45Ser Val Asn Gly Ser Thr Thr Thr Phe Ile Asp
Lys Asn Ile Val Tyr 50 55 60Ser Gln Thr Tyr Tyr Tyr Lys Val Ser Ala
Val Asn Asn Ile Val Glu65 70 75 80Gly Pro Lys Ser Asn Thr Ala Ser
Ala Thr Pro Thr Ser Ser 85 90387PRTArtificialSynthetic construct
3Pro Pro Lys Pro Gln Ile Ala Ser Ile Ala Ser Gly Asn Glu Thr Ile1 5
10 15Thr Val Lys Trp Tyr Asp Thr Asn Ala Ser Gly Tyr Tyr Ile Thr
Tyr 20 25 30Trp Ser Asn Phe Ser Gln Lys Val Thr Ile Asn Val Gly Asn
Val Thr 35 40 45Ser Tyr Thr Ile Lys His Leu Lys Asp Gly Val Thr Tyr
Tyr Ile Gln 50 55 60Ile Val Pro Tyr Asn Ser Leu Gly Asn Gly Thr Pro
Ser Asp Ile Ile65 70 75 80Ser Ala Thr Pro Ser Ser Val
85486PRTArtificialSynthetic construct 4Pro Asn Pro Pro Ile Ile Lys
Val Lys Ile Gly Asn Leu Asn Ala Thr1 5 10 15Leu Thr Trp Tyr Asp Thr
Phe Asn Gly Gly Tyr Pro Ile Glu Gly Tyr 20 25 30Tyr Leu Tyr Val Asn
Gly Lys Gly Ile Asn Val Gly Asn Ile Thr Ser 35 40 45Tyr Val Leu Thr
Asn Leu Thr Ala Gly Glu Leu Tyr Thr Ile Glu Leu 50 55 60Ile Ala Tyr
Asn Lys Ile Gly Asn Ser Ser Ile Ser Ser Val Ser Phe65 70 75 80Ile
Ala Ala Ser Lys Ala 855101PRTArtificialSynthetic construct 5Ala Ala
Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser1 5 10 15Trp
Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly 20 25
30Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser
35 40 45Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr 50 55 60Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala
Ser Ser65 70 75 80Lys Pro Ile Ser Ile Asn Tyr Arg Thr Gly Gly Gly
Thr Leu Glu His 85 90 95His His His His His
100687PRTArtificialSynthetic construct 6Leu Glu Val Val Ala Ala Thr
Pro Thr Ser Leu Leu Ile Ser Trp Asp1 5 10 15Ala Pro Ala Val Thr Val
Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr 20 25 30Gly Gly Asn Ser Pro
Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser 35 40 45Thr Ala Thr Ile
Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr 50 55 60Val Tyr Ala
Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro65 70 75 80Ile
Ser Ile Asn Tyr Arg Thr 85796PRTArtificialSynthetic construct 7Ala
Ala Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg1 5 10
15Trp Ser Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser
20 25 30Pro Ser Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr
Ala 35 40 45Asn Ser Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr
Asn Ile 50 55 60Thr Ile Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro
Val Val Ile65 70 75 80Gln Gln Glu Thr Gly Gly Gly Thr Leu Glu His
His His His His His 85 90 95882PRTArtificialSynthetic construct
8Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser1 5
10 15Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro
Ser 20 25 30Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala
Asn Ser 35 40 45Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn
Ile Thr Ile 50 55 60Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val
Val Ile Gln Gln65 70 75 80Glu Thr994PRTArtificialSynthetic
construct 9Ala Ala Pro Tyr Asn Thr Glu Val Thr Glu Thr Thr Ile Val
Ile Thr1 5 10 15Trp Thr Pro Ala Pro Arg Ile Gly Phe Lys Leu Gly Val
Arg Pro Ser 20 25 30Gln Gly Gly Glu Ala Pro Arg Glu Val Thr Ser Asp
Ser Gly Ser Ile 35 40 45Val Val Ser Gly Leu Thr Pro Gly Val Glu Tyr
Val Tyr Thr Ile Gln 50 55 60Val Leu Arg Asp Gly Gln Glu Arg Asp Ala
Pro Ile Val Asn Lys Val65 70 75 80Val Thr Gly Gly Gly Thr Leu Glu
His His His His His His 85 901080PRTArtificialSynthetic construct
10Pro Tyr Asn Thr Glu Val Thr Glu Thr Thr Ile Val Ile Thr Trp Thr1
5 10 15Pro Ala Pro Arg Ile Gly Phe Lys Leu Gly Val Arg Pro Ser Gln
Gly 20 25 30Gly Glu Ala Pro Arg Glu Val Thr Ser Asp Ser Gly Ser Ile
Val Val 35 40 45Ser Gly Leu Thr Pro Gly Val Glu Tyr Val Tyr Thr Ile
Gln Val Leu 50 55 60Arg Asp Gly Gln Glu Arg Asp Ala Pro Ile Val Asn
Lys Val Val Thr65 70 75 8011116PRTArtificialSynthetic construct
11Ala Ala Pro Pro Ile Ala Leu Asn Trp Thr Leu Leu Asn Val Ser Leu1
5 10 15Thr Gly Ile His Ala Asp Ile Gln Val Arg Trp Glu Ala Pro Arg
Asn 20 25 30Ala Asp Ile Gln Lys Gly Trp Met Val Leu Glu Tyr Glu Leu
Gln Tyr 35 40 45Lys Glu Val Asn Glu Thr Lys Trp Lys Met Met Asp Pro
Ile Leu Thr 50 55 60Thr Ser Val Pro Val Tyr Ser Leu Lys Val Asp Lys
Glu Tyr Glu Val65 70 75 80Arg Val Arg Ser Lys Gln Arg Asn Ser Gly
Asn Tyr Gly Glu Phe Ser 85 90 95Glu Val Leu Tyr Val Thr Leu Pro Gly
Gly Gly Thr Leu Glu His His 100 105 110His His His His
11512102PRTArtificialSynthetic construct 12Pro Pro Ile Ala Leu Asn
Trp Thr Leu Leu Asn Val Ser Leu Thr Gly1 5 10 15Ile His Ala Asp Ile
Gln Val Arg Trp Glu Ala Pro Arg Asn Ala Asp 20 25 30Ile Gln Lys Gly
Trp Met Val Leu Glu Tyr Glu Leu Gln Tyr Lys Glu 35 40 45Val Asn Glu
Thr Lys Trp Lys Met Met Asp Pro Ile Leu Thr Thr Ser 50 55 60Val Pro
Val Tyr Ser Leu Lys Val Asp Lys Glu Tyr Glu Val Arg Val65 70 75
80Arg Ser Lys Gln Arg Asn Ser Gly Asn Tyr Gly Glu Phe Ser Glu Val
85 90 95Leu Tyr Val Thr Leu Pro 10013110PRTArtificialSynthetic
construct 13Ala Ala Pro Pro Ser Leu Asn Val Thr Lys Asp Gly Asp Ser
Tyr Ser1 5 10 15Leu Arg Trp Glu Thr Met Lys Met Arg Tyr Glu His Ile
Asp His Thr 20 25 30Phe Glu Ile Gln Tyr Arg Lys Asp Thr Ala Thr Trp
Lys Asp Ser Lys 35 40 45Thr Glu Thr Leu Gln Asn Ala His Ser Met Ala
Leu Pro Ala Leu Glu 50 55 60Pro Ser Thr Arg Tyr Trp Ala Arg Val Arg
Val Arg Thr Ser Arg Thr65 70 75 80Gly Tyr Asn Gly Ile Trp Ser Glu
Trp Ser Glu Ala Arg Ser Trp Asp 85 90 95Thr Glu Gly Gly Gly Thr Leu
Glu His His His His His His 100 105 1101496PRTArtificialSynthetic
construct 14Pro Pro Ser Leu Asn Val Thr Lys Asp Gly Asp Ser Tyr Ser
Leu Arg1 5 10 15Trp Glu Thr Met Lys Met Arg Tyr Glu His Ile Asp His
Thr Phe Glu 20 25 30Ile Gln Tyr Arg Lys Asp Thr Ala Thr Trp Lys Asp
Ser Lys Thr Glu 35 40 45Thr Leu Gln Asn Ala His Ser Met Ala Leu Pro
Ala Leu Glu Pro Ser 50 55 60Thr Arg Tyr Trp Ala Arg Val Arg Val Arg
Thr Ser Arg Thr Gly Tyr65 70 75 80Asn Gly Ile Trp Ser Glu Trp Ser
Glu Ala Arg Ser Trp Asp Thr Glu 85 90 9515103PRTArtificialSynthetic
construct 15Ala Ala Pro Pro Val Asn Phe Thr Ile Lys Val Thr Gly Leu
Ala Gln1 5 10 15Val Leu Leu Gln Trp Lys Pro Asn Pro Asp Gln Glu Gln
Arg Asn Val 20 25 30Asn Leu Glu Tyr Gln Val Lys Ile Asn Ala Pro Lys
Glu Asp Asp Tyr 35 40 45Glu Thr Arg Ile Thr Glu Ser Lys Leu Val Thr
Ile Leu His Lys Gly 50 55 60Phe Ser Ala Ser Val Arg Thr Ile Leu Gln
Asn Asp His Ser Leu Leu65 70 75 80Ala Ser Ser Trp Ala Ser Ala Glu
Leu His Ala Gly Gly Gly Thr Leu 85 90 95Glu His His His His His His
1001689PRTArtificialSynthetic construct 16Pro Pro Val Asn Phe Thr
Ile Lys Val Thr Gly Leu Ala Gln Val Leu1 5 10 15Leu Gln Trp Lys Pro
Asn Pro Asp Gln Glu Gln Arg Asn Val Asn Leu 20 25 30Glu Tyr Gln Val
Lys Ile Asn Ala Pro Lys Glu Asp Asp Tyr Glu Thr 35 40 45Arg Ile Thr
Glu Ser Lys Leu Val Thr Ile Leu His Lys Gly Phe Ser 50 55 60Ala Ser
Val Arg Thr Ile Leu Gln Asn Asp His Ser Leu Leu Ala Ser65 70 75
80Ser Trp Ala Ser Ala Glu Leu His Ala 8517105PRTArtificialSynthetic
construct 17Ala Ala Leu Ser Val Thr Asp Val Thr Thr Ser Ser Leu Arg
Leu Asn1 5 10 15Trp Glu Ala Pro Pro Gly Ala Phe Asp Ser Phe Leu Leu
Arg Phe Gly 20 25 30Val Pro Ser Pro Ser Thr Leu Glu Pro His Pro Arg
Pro Leu Leu Gln 35 40 45Arg Glu Leu Met Val Pro Gly Thr Arg His Ser
Ala Val Leu Arg Asp 50 55 60Leu Arg Ser Gly Thr Leu Tyr Ser Leu Thr
Leu Tyr Gly Leu Arg Gly65 70 75 80Pro His Lys Ala Asp Ser Ile Gln
Gly Thr Ala Arg Thr Gly Gly Gly 85 90 95Thr Leu Glu His His His His
His His 100 1051891PRTArtificialSynthetic construct 18Leu Ser Val
Thr Asp Val Thr Thr Ser Ser Leu Arg Leu Asn Trp Glu1 5 10 15Ala Pro
Pro Gly Ala Phe Asp Ser Phe Leu Leu Arg Phe Gly Val Pro 20 25 30Ser
Pro Ser Thr Leu Glu Pro His Pro Arg Pro Leu Leu Gln Arg Glu 35 40
45Leu Met Val Pro Gly Thr Arg His Ser Ala Val Leu Arg Asp Leu Arg
50 55 60Ser Gly Thr Leu Tyr Ser Leu Thr Leu Tyr Gly Leu Arg Gly Pro
His65 70 75 80Lys Ala Asp Ser Ile Gln Gly Thr Ala Arg Thr 85
901995PRTArtificialSynthetic construct 19Ala Ala Leu Arg Ala Leu
Asn Leu Thr Glu Gly Phe Ala Val Leu His1 5 10 15Trp Lys Pro Pro Gln
Asn Pro Val Asp Thr Tyr Asp Ile Gln Val Thr 20 25 30Ala Pro Gly Ala
Pro Pro Leu Gln Ala Glu Thr Pro Gly Ser Ala Val 35 40 45Asp Tyr Pro
Leu His Asp Leu Val Leu His Thr Asn Tyr Thr Ala Thr 50 55 60Val Arg
Gly Leu Arg Gly Pro Asn Leu Thr Ser Pro Ala Ser Ile Thr65 70 75
80Phe Thr Thr Gly Gly Gly Thr Leu Glu His His His His His His 85 90
952081PRTArtificialSynthetic construct 20Leu Arg Ala Leu Asn Leu
Thr Glu Gly Phe Ala Val Leu His Trp Lys1 5 10 15Pro Pro Gln Asn Pro
Val Asp Thr Tyr Asp Ile Gln Val Thr Ala Pro 20 25 30Gly Ala Pro Pro
Leu Gln Ala Glu Thr Pro Gly Ser Ala Val Asp Tyr 35 40 45Pro Leu His
Asp Leu Val Leu His Thr Asn Tyr Thr Ala Thr Val Arg 50 55 60Gly Leu
Arg Gly Pro Asn Leu Thr Ser Pro Ala Ser Ile Thr Phe Thr65 70 75
80Thr 2195PRTArtificialSynthetic consrtuct 21Ala Ala Leu Glu Ala
Lys Glu Val Thr Pro Arg Thr Ala Leu Leu Thr1 5 10 15Trp Thr Glu Pro
Pro Val Arg Pro Ala Gly Tyr Leu Leu Ser Phe His 20 25 30Thr Pro Gly
Gly Gln Thr Gln Glu Ile Leu Leu Pro Gly Gly Ile Thr 35 40 45Ser His
Gln Leu Leu Gly Leu Phe Pro Ser Thr Ser Tyr Asn Ala Arg 50 55 60Leu
Gln Ala Met Trp Gly Gln Ser Leu Leu Pro Pro Val Ser Thr Ser65 70 75
80Phe Thr Thr Gly Gly Gly Thr Leu Glu His His His His His His 85 90
952281PRTArtificialSynthetic construct 22Leu Glu Ala Lys Glu Val
Thr Pro Arg Thr Ala Leu Leu Thr Trp Thr1 5 10 15Glu Pro Pro Val Arg
Pro Ala Gly Tyr Leu Leu Ser Phe His Thr Pro 20 25 30Gly Gly Gln Thr
Gln Glu Ile Leu Leu Pro Gly Gly Ile Thr Ser His 35 40 45Gln Leu Leu
Gly Leu Phe Pro Ser Thr Ser Tyr Asn Ala Arg Leu Gln 50 55 60Ala Met
Trp Gly Gln Ser Leu Leu Pro Pro Val Ser Thr Ser Phe Thr65 70 75
80Thr 2397PRTArtificialSynthetic construct 23Ala Ala Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr1 5 10 15Trp Phe Lys Pro
Leu Ala Glu Ile Asp Gly Ile Glu Leu Thr Tyr Gly 20 25 30Ile Lys Asp
Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp 35 40 45Glu Asn
Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60Val
Ser Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys65 70 75
80Glu Thr Phe Thr Thr Gly Gly Gly Thr Leu Glu His His His His His
85 90 95His 2483PRTArtificialSynthetic construct 24Ile Glu Val Lys
Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe1 5 10 15Lys Pro Leu
Ala Glu Ile Asp Gly Ile Glu Leu Thr Tyr Gly Ile Lys 20 25 30Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn 35 40 45Gln
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55
60Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr65
70 75 80Phe Thr Thr25107PRTArtificialSynthetic construct 25Ala Ala
Pro Lys Phe Thr Lys Cys Arg Ser Pro Glu Arg Glu Thr Phe1 5 10 15Ser
Cys His Trp Thr Asp Glu Val His His Gly Thr Lys Asn Leu Gly 20 25
30Pro Ile Gln Leu Phe Tyr Thr Arg Arg Asn Thr Gln Glu Trp Thr Gln
35 40 45Glu Trp Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser
Cys 50 55 60Tyr Phe Asn Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys
Ile Lys65 70 75 80Leu Thr Ser Asn Gly Gly Thr Val Asp Glu Lys Cys
Phe Ser Val Gly 85 90 95Gly Gly Thr Leu Glu His His His His His His
100 1052693PRTArtificialSynthetic construct 26Pro Lys
Phe Thr Lys Cys Arg Ser Pro Glu Arg Glu Thr Phe Ser Cys1 5 10 15His
Trp Thr Asp Glu Val His His Gly Thr Lys Asn Leu Gly Pro Ile 20 25
30Gln Leu Phe Tyr Thr Arg Arg Asn Thr Gln Glu Trp Thr Gln Glu Trp
35 40 45Lys Glu Cys Pro Asp Tyr Val Ser Ala Gly Glu Asn Ser Cys Tyr
Phe 50 55 60Asn Ser Ser Phe Thr Ser Ile Trp Ile Pro Tyr Cys Ile Lys
Leu Thr65 70 75 80Ser Asn Gly Gly Thr Val Asp Glu Lys Cys Phe Ser
Val 85 9027111PRTArtificialSynthetic construct 27Ala Ala Pro Ser
Gly Phe Pro Gln Asn Leu His Val Thr Gly Leu Thr1 5 10 15Thr Ser Thr
Thr Glu Leu Ala Trp Asp Pro Pro Val Leu Ala Glu Arg 20 25 30Asn Gly
Arg Ile Ile Ser Tyr Thr Val Val Phe Arg Asp Ile Asn Ser 35 40 45Gln
Gln Glu Leu Gln Asn Ile Thr Thr Asp Thr Arg Phe Thr Leu Thr 50 55
60Gly Leu Lys Pro Asp Thr Thr Tyr Asp Ile Lys Val Arg Ala Trp Thr65
70 75 80Ser Lys Gly Ser Gly Pro Leu Ser Pro Ser Ile Gln Ser Arg Thr
Met 85 90 95Pro Val Glu Gly Gly Gly Thr Leu Glu His His His His His
His 100 105 1102897PRTArtificialSynthetic construct 28Pro Ser Gly
Phe Pro Gln Asn Leu His Val Thr Gly Leu Thr Thr Ser1 5 10 15Thr Thr
Glu Leu Ala Trp Asp Pro Pro Val Leu Ala Glu Arg Asn Gly 20 25 30Arg
Ile Ile Ser Tyr Thr Val Val Phe Arg Asp Ile Asn Ser Gln Gln 35 40
45Glu Leu Gln Asn Ile Thr Thr Asp Thr Arg Phe Thr Leu Thr Gly Leu
50 55 60Lys Pro Asp Thr Thr Tyr Asp Ile Lys Val Arg Ala Trp Thr Ser
Lys65 70 75 80Gly Ser Gly Pro Leu Ser Pro Ser Ile Gln Ser Arg Thr
Met Pro Val 85 90 95Glu29108PRTArtificialSynthetic construct 29Ala
Ala Pro Lys Pro Pro Ile Asp Leu Val Val Thr Glu Thr Thr Ala1 5 10
15Thr Ser Val Thr Leu Thr Trp Asp Ser Gly Asn Ser Glu Pro Val Thr
20 25 30Tyr Tyr Gly Ile Gln Tyr Arg Ala Ala Gly Thr Glu Gly Pro Phe
Gln 35 40 45Glu Val Asp Gly Val Ala Thr Thr Arg Tyr Ser Ile Gly Gly
Leu Ser 50 55 60Pro Phe Ser Glu Tyr Ala Phe Arg Val Leu Ala Val Asn
Ser Ile Gly65 70 75 80Arg Gly Pro Pro Ser Glu Ala Val Arg Ala Arg
Thr Gly Glu Gln Ala 85 90 95Gly Gly Gly Thr Leu Glu His His His His
His His 100 1053092PRTArtificialSynthetic construct 30Pro Lys Pro
Pro Ile Asp Leu Val Val Thr Glu Thr Thr Ala Thr Ser1 5 10 15Val Thr
Leu Thr Trp Asp Ser Gly Asn Ser Glu Pro Val Thr Tyr Tyr 20 25 30Gly
Ile Gln Tyr Arg Ala Ala Gly Thr Glu Gly Pro Phe Gln Glu Val 35 40
45Asp Gly Val Ala Thr Thr Arg Tyr Ser Ile Gly Gly Leu Ser Pro Phe
50 55 60Ser Glu Tyr Ala Phe Arg Val Leu Ala Val Asn Ser Ile Gly Arg
Gly65 70 75 80Pro Pro Ser Glu Ala Val Arg Ala Arg Thr Gly Glu 85
9031101PRTArtificialSynthetic construct 31Ala Ala Leu Ser Pro Pro
Arg Asn Leu Arg Ile Ser Asn Val Gly Ser1 5 10 15Asn Ser Ala Arg Leu
Thr Trp Asp Pro Thr Ser Arg Gln Ile Asn Gly 20 25 30Tyr Arg Ile Val
Tyr Asn Asn Ala Asp Gly Thr Glu Ile Asn Glu Val 35 40 45Glu Val Asp
Pro Ile Thr Thr Phe Pro Leu Lys Gly Leu Thr Pro Leu 50 55 60Thr Glu
Tyr Thr Ile Ala Ile Phe Ser Ile Tyr Asp Glu Gly Gln Ser65 70 75
80Glu Pro Leu Thr Gly Val Phe Thr Thr Gly Gly Gly Thr Leu Glu His
85 90 95His His His His His 1003287PRTArtificialSythetic construct
32Leu Ser Pro Pro Arg Asn Leu Arg Ile Ser Asn Val Gly Ser Asn Ser1
5 10 15Ala Arg Leu Thr Trp Asp Pro Thr Ser Arg Gln Ile Asn Gly Tyr
Arg 20 25 30Ile Val Tyr Asn Asn Ala Asp Gly Thr Glu Ile Asn Glu Val
Glu Val 35 40 45Asp Pro Ile Thr Thr Phe Pro Leu Lys Gly Leu Thr Pro
Leu Thr Glu 50 55 60Tyr Thr Ile Ala Ile Phe Ser Ile Tyr Asp Glu Gly
Gln Ser Glu Pro65 70 75 80Leu Thr Gly Val Phe Thr Thr
8533285DNAArtificialSynthetic construct 33gccatggccg ccctggaagt
ggtggcggcg accccgacca gcctgctgat tagctgggat 60gcgccggcgg tgaccgtgcg
ctattatcgt attacctatg gcgaaaccgg cggcaatagc 120ccggtgcagg
aatttaccgt gccgggcagc aaaagcaccg cgaccattag cggcctgaaa
180ccgggcgtgg attataccat taccgtgtat gcggtgaccg gccgtggcga
tagcccggcg 240agcagcaaac cgattagcat taactatcgt accggtggcg gtacc
28534261DNAArtificialSynthetic construct 34ctggaagtgg tggcggcgac
cccgaccagc ctgctgatta gctgggatgc gccggcggtg 60accgtgcgct attatcgtat
tacctatggc gaaaccggcg gcaatagccc ggtgcaggaa 120tttaccgtgc
cgggcagcaa aagcaccgcg accattagcg gcctgaaacc gggcgtggat
180tataccatta ccgtgtatgc ggtgaccggc cgtggcgata gcccggcgag
cagcaaaccg 240attagcatta actatcgtac c
26135270DNAArtificialSynthetic construct 35gccatggccg ccccgaccgt
ggatcaggtg gatgatacca gcattgtggt gcgctggagc 60cgtccgcagg cgccgattac
cggctatcgt attgtgtata gcccgagcgt ggaaggcagc 120agcaccgaac
tgaacctgcc ggaaaccgcg aatagcgtga ccctgagcga tctgcagccg
180ggcgtgcagt ataacattac catttatgcg gtggaagaaa accaggaaag
caccccggtg 240gtgattcagc aggaaaccgg tggcggtacc
27036246DNAArtificialSynthetic construct 36ccgaccgtgg atcaggtgga
tgataccagc attgtggtgc gctggagccg tccgcaggcg 60ccgattaccg gctatcgtat
tgtgtatagc ccgagcgtgg aaggcagcag caccgaactg 120aacctgccgg
aaaccgcgaa tagcgtgacc ctgagcgatc tgcagccggg cgtgcagtat
180aacattacca tttatgcggt ggaagaaaac caggaaagca ccccggtggt
gattcagcag 240gaaacc 24637264DNAArtificialSynthetic construct
37gccatggccg ccccgtataa caccgaagtg accgaaacca ccattgtgat tacctggacc
60ccggcgccgc gtattggctt taaactgggc gtgcgtccga gccagggcgg tgaagcgccg
120cgcgaagtga ccagcgatag cggcagcatt gtggtgagcg gcctgacccc
gggcgtggaa 180tatgtgtata ccattcaggt gctgcgtgat ggccaggaac
gtgatgcgcc gattgtgaac 240aaagtggtga ccggtggcgg tacc
26438240DNAArtificialSynthetic construct 38ccgtataaca ccgaagtgac
cgaaaccacc attgtgatta cctggacccc ggcgccgcgt 60attggcttta aactgggcgt
gcgtccgagc cagggcggtg aagcgccgcg cgaagtgacc 120agcgatagcg
gcagcattgt ggtgagcggc ctgaccccgg gcgtggaata tgtgtatacc
180attcaggtgc tgcgtgatgg ccaggaacgt gatgcgccga ttgtgaacaa
agtggtgacc 24039330DNAArtificialSynthetic construct 39gccatggccg
ccccgccgat cgctctgaat tggaccctgc tgaatgtttc gctgaccggt 60attcatgccg
atattcaggt gcgttgggaa gcgccgcgta acgccgatat tcagaaaggc
120tggatggtgc tggaatatga actgcagtat aaagaagtga atgaaaccaa
atggaaaatg 180atggacccga ttctgaccac cagcgtgccg gtgtacagcc
tgaaagtgga taaagaatac 240gaagtccgtg tgcgttctaa acagcgtaat
agcggcaatt atggtgaatt tagtgaagtc 300ctgtatgtta ccctgccggg
tggcggtacc 33040306DNAArtificialSynthetic construct 40ccgccgatcg
ctctgaattg gaccctgctg aatgtttcgc tgaccggtat tcatgccgat 60attcaggtgc
gttgggaagc gccgcgtaac gccgatattc agaaaggctg gatggtgctg
120gaatatgaac tgcagtataa agaagtgaat gaaaccaaat ggaaaatgat
ggacccgatt 180ctgaccacca gcgtgccggt gtacagcctg aaagtggata
aagaatacga agtccgtgtg 240cgttctaaac agcgtaatag cggcaattat
ggtgaattta gtgaagtcct gtatgttacc 300ctgccg
30641312DNAArtificialSynthetic construct 41gccatggccg ccccgccgag
cctgaacgtg accaaagatg gcgatagcta tagcctgcgc 60tgggaaacca tgaaaatgcg
ctatgaacat attgatcata cctttgaaat tcagtatcgc 120aaagataccg
cgacctggaa agatagcaaa accgaaaccc tgcagaacgc gcatagcatg
180gcgctgccgg cgctggaacc gagcacccgt tattgggcgc gtgtgcgtgt
gcgtaccagc 240cgtaccggct ataatggcat ttggagcgaa tggagcgaag
cgcgtagctg ggataccgaa 300ggtggcggta cc
31242291DNAArtificialSynthetic construct 42gccatggccg ccccgccggt
gaactttacc attaaagtga ccggcctggc gcaggtgctg 60ctgcagtgga aaccgaaccc
ggatcaggaa cagcgtaacg tgaacctgga atatcaggtg 120aaaattaacg
cgccgaaaga agatgattat gaaacccgca ttaccgaaag caaactggtg
180accattctgc ataaaggctt tagcgcgagc gtgcgtacca ttctgcagaa
cgatcatagc 240ctgctggcga gcagctgggc gagcgcggaa ctgcatgcgg
gtggcggtac c 29143267DNAArtificialSynthetic construct 43ccgccggtga
actttaccat taaagtgacc ggcctggcgc aggtgctgct gcagtggaaa 60ccgaacccgg
atcaggaaca gcgtaacgtg aacctggaat atcaggtgaa aattaacgcg
120ccgaaagaag atgattatga aacccgcatt accgaaagca aactggtgac
cattctgcat 180aaaggcttta gcgcgagcgt gcgtaccatt ctgcagaacg
atcatagcct gctggcgagc 240agctgggcga gcgcggaact gcatgcg
26744297DNAArtificialSynthetic construct 44gccatggccg ccctgagcgt
gaccgatgtg accaccagca gcctgcgtct gaactgggaa 60gcgccgccgg gcgcgtttga
tagctttctg ctgcgttttg gcgtgccgag cccgagcacc 120ctggaaccgc
atccgcgtcc gctgctgcag cgtgaactga tggtgccggg cacccgtcat
180agcgcggtgc tgcgtgatct gcgtagcggc accctgtata gcctgaccct
gtatggcctg 240cgtggcccgc ataaagcgga tagcattcag ggcaccgcgc
gtaccggtgg cggtacc 29745273DNAArtificialSynthetic construct
45ctgagcgtga ccgatgtgac caccagcagc ctgcgtctga actgggaagc gccgccgggc
60gcgtttgata gctttctgct gcgttttggc gtgccgagcc cgagcaccct ggaaccgcat
120ccgcgtccgc tgctgcagcg tgaactgatg gtgccgggca cccgtcatag
cgcggtgctg 180cgtgatctgc gtagcggcac cctgtatagc ctgaccctgt
atggcctgcg tggcccgcat 240aaagcggata gcattcaggg caccgcgcgt acc
27346267DNAArtificialSynthetic construct 46gccatggccg ccctgcgcgc
gctgaacctg accgaaggct ttgcggtgct gcattggaaa 60ccgccgcaga acccggtgga
tacctatgat attcaggtga ccgcgccggg cgcgccgccg 120ctgcaggcgg
aaaccccggg cagcgcggtg gattatccgc tgcatgatct ggtgctgcat
180accaactata ccgcgaccgt gcgtggcctg cgcggcccga atctgaccag
cccggcgagc 240attaccttta ccaccggtgg cggtacc
26747243DNAArtificialSynthetic construct 47ctgcgcgcgc tgaacctgac
cgaaggcttt gcggtgctgc attggaaacc gccgcagaac 60ccggtggata cctatgatat
tcaggtgacc gcgccgggcg cgccgccgct gcaggcggaa 120accccgggca
gcgcggtgga ttatccgctg catgatctgg tgctgcatac caactatacc
180gcgaccgtgc gtggcctgcg cggcccgaat ctgaccagcc cggcgagcat
tacctttacc 240acc 24348267DNAArtificialSynthetic construct
48gccatggccg ccctggaagc gaaagaagtg accccgcgta ccgcgctgct gacctggacc
60gaaccgccgg tgcgcccggc gggttatctg ctgagctttc ataccccggg cggccagacc
120caggaaattc tgctgccggg cggcattacc agccatcagc tgctgggcct
gtttccgagc 180accagctata acgcgcgtct gcaggcgatg tggggccaga
gcctgctgcc gccggtgagc 240accagcttta ccaccggtgg cggtacc
26749243DNAArtificialSynthetic construct 49ctggaagcga aagaagtgac
cccgcgtacc gcgctgctga cctggaccga accgccggtg 60cgcccggcgg gttatctgct
gagctttcat accccgggcg gccagaccca ggaaattctg 120ctgccgggcg
gcattaccag ccatcagctg ctgggcctgt ttccgagcac cagctataac
180gcgcgtctgc aggcgatgtg gggccagagc ctgctgccgc cggtgagcac
cagctttacc 240acc 24350273DNAArtificialSynthetic construct
50gccatggccg ccattgaagt gaaagatgtg accgatacca ccgcgctgat tacctggttt
60aaaccgctgg cggaaattga tggcattgaa ctgacctatg gcattaaaga tgtgccgggc
120gatcgcacca ccattgatct gaccgaagat gaaaaccagt atagcattgg
caacctgaaa 180ccggataccg aatatgaagt gagcctgatt agccgtcgtg
gcgatatgag cagcaacccg 240gcgaaagaaa cctttaccac cggtggcggt acc
27351249DNAArtificialSynthetic construct 51attgaagtga aagatgtgac
cgataccacc gcgctgatta cctggtttaa accgctggcg 60gaaattgatg gcattgaact
gacctatggc attaaagatg tgccgggcga tcgcaccacc 120attgatctga
ccgaagatga aaaccagtat agcattggca acctgaaacc ggataccgaa
180tatgaagtga gcctgattag ccgtcgtggc gatatgagca gcaacccggc
gaaagaaacc 240tttaccacc 24952303DNAArtificialSynthetic construct
52gccatggccg ccccgaaatt taccaaatgc cgtagcccgg aacgcgaaac ctttagctgc
60cattggaccg atgaagttca tcatggcacc aaaaatctgg gcccgattca gctgttttat
120acccgccgta atacccagga atggacccag gaatggaaag aatgcccgga
ttatgttagc 180gcgggcgaaa acagctgcta ttttaacagc agctttacca
gcatttggat tccgtattgc 240attaaactga ccagcaacgg tggcaccgtt
gatgaaaaat gctttagcgt gggtggcggt 300acc
30353279DNAArtificialSynthetic construct 53ccgaaattta ccaaatgccg
tagcccggaa cgcgaaacct ttagctgcca ttggaccgat 60gaagttcatc atggcaccaa
aaatctgggc ccgattcagc tgttttatac ccgccgtaat 120acccaggaat
ggacccagga atggaaagaa tgcccggatt atgttagcgc gggcgaaaac
180agctgctatt ttaacagcag ctttaccagc atttggattc cgtattgcat
taaactgacc 240agcaacggtg gcaccgttga tgaaaaatgc tttagcgtg
27954315DNAArtificialSynthetic construct 54gccatggcag ccccgtctgg
ttttccgcag aatctgcatg tgaccggcct gaccaccagc 60accaccgaac tggcgtggga
tccgccggtg ctggcggaac gcaacggccg tattattagc 120tataccgtgg
tgtttcgtga tattaacagc cagcaggaac tgcagaacat taccaccgat
180acccgcttta ccctgaccgg tctgaaaccg gataccacct atgatattaa
agtgcgcgcc 240tggaccagca aaggcagcgg cccgctgagc ccgagcattc
agagccgcac catgccggtg 300gaaggtggcg gtacc
31555291DNAArtificialSynthetic construct 55ccgtctggtt ttccgcagaa
tctgcatgtg accggcctga ccaccagcac caccgaactg 60gcgtgggatc cgccggtgct
ggcggaacgc aacggccgta ttattagcta taccgtggtg 120tttcgtgata
ttaacagcca gcaggaactg cagaacatta ccaccgatac ccgctttacc
180ctgaccggtc tgaaaccgga taccacctat gatattaaag tgcgcgcctg
gaccagcaaa 240ggcagcggcc cgctgagccc gagcattcag agccgcacca
tgccggtgga a 29156306DNAArtificialSynthetic construct 56gccatggccg
ccccgaaacc gccgattgat ctggtggtta ccgaaaccac cgcgaccagc 60gtgaccctga
cctgggatag cggcaatagc gaaccggtga cctattatgg tattcagtat
120cgcgcggcgg gcaccgaagg tccgtttcag gaagtggatg gcgtggcgac
cacccgttat 180agcattggcg gtctgagccc gtttagcgaa tatgcgtttc
gcgtgctggc ggttaatagc 240attggccgcg gtccgccgag cgaagcggtg
cgtgcgcgca ccggcgaaca ggcgggtggc 300ggtacc
30657282DNAArtificialSynthetic construct 57ccgaaaccgc cgattgatct
ggtggttacc gaaaccaccg cgaccagcgt gaccctgacc 60tgggatagcg gcaatagcga
accggtgacc tattatggta ttcagtatcg cgcggcgggc 120accgaaggtc
cgtttcagga agtggatggc gtggcgacca cccgttatag cattggcggt
180ctgagcccgt ttagcgaata tgcgtttcgc gtgctggcgg ttaatagcat
tggccgcggt 240ccgccgagcg aagcggtgcg tgcgcgcacc ggcgaacagg cg
28258285DNAArtificialSynthetic construct 58gccatggccg ccctgagccc
gccgcgtaac ctgcgcatta gcaacgtggg tagcaatagc 60gcgcgcctga cctgggatcc
gaccagccgc cagattaatg gctatcgcat tgtgtataac 120aacgccgatg
gcaccgaaat taacgaagtg gaagtggatc cgattaccac ctttccgctg
180aaaggcctga ccccgctgac cgaatatacc attgcgattt ttagcattta
tgatgaaggt 240cagagcgaac cgctgaccgg tgtgtttacc accggtggcg gtacc
28559261DNAArtificialSynthetic construct 59ctgagcccgc cgcgtaacct
gcgcattagc aacgtgggta gcaatagcgc gcgcctgacc 60tgggatccga ccagccgcca
gattaatggc tatcgcattg tgtataacaa cgccgatggc 120accgaaatta
acgaagtgga agtggatccg attaccacct ttccgctgaa aggcctgacc
180ccgctgaccg aatataccat tgcgattttt agcatttatg atgaaggtca
gagcgaaccg 240ctgaccggtg tgtttaccac c 2616097PRTArtificialSynthetic
construct 60Ala Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr1 5 10 15Trp Phe Lys Pro Leu Ala Glu Ile Asp Gly Ile Glu Leu
Thr Tyr Gly 20 25 30Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp
Leu Thr Glu Asp 35 40 45Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro
Asp Thr Glu Tyr Glu 50 55 60Val Ser Leu Ile Ser Arg Arg Gly Asp Met
Ser Ser Asn Pro Ala Lys65 70 75 80Glu Thr Phe Thr Thr Gly Gly Gly
Thr Leu Glu His His His His His 85 90
95His6183PRTArtificialSynthetic construct 61Ile Glu Val Lys Asp Val
Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe1 5
10 15Lys Pro Leu Ala Glu Ile Asp Gly Ile Glu Leu Thr Tyr Gly Ile
Lys 20 25 30Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp
Glu Asn 35 40 45Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr
Glu Val Ser 50 55 60Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn Pro
Ala Lys Glu Thr65 70 75 80Phe Thr Thr6297PRTArtificialSynthetic
construct 62Ala Ala Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu
Ile Thr1 5 10 15Trp Ser Pro Pro Ser Val Leu Val Gly Tyr Thr Ile Glu
Leu Thr Tyr 20 25 30Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile
Asp Leu Thr Glu 35 40 45Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys
Pro Asp Thr Glu Tyr 50 55 60Glu Val Ser Leu Ile Ser Val Thr Glu Phe
Gly Arg Arg Arg Ser Lys65 70 75 80Glu Thr Phe Thr Thr Gly Gly Gly
Thr Leu Glu His His His His His 85 90
95His6383PRTArtificialSynthetic construct 63Ile Glu Val Lys Asp Val
Thr Asp Thr Thr Ala Leu Ile Thr Trp Ser1 5 10 15Pro Pro Ser Val Leu
Val Gly Tyr Thr Ile Glu Leu Thr Tyr Gly Ile 20 25 30Lys Asp Val Pro
Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu 35 40 45Asn Gln Tyr
Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val 50 55 60Ser Leu
Ile Ser Val Thr Glu Phe Gly Arg Arg Arg Ser Lys Glu Thr65 70 75
80Phe Thr Thr6483PRTArtificialSynthetic construct 64Ile Glu Cys Lys
Asp Val Thr Asp Thr Thr Ala Leu Cys Thr Trp Phe1 5 10 15Lys Pro Leu
Ala Glu Ile Asp Gly Ile Glu Leu Thr Tyr Gly Ile Lys 20 25 30Asp Val
Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn 35 40 45Gln
Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser 50 55
60Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr65
70 75 80Phe Thr Thr6583PRTArtificialSynthetic construct 65Ile Glu
Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe1 5 10 15Lys
Pro Leu Ala Glu Ile Asp Gly Ile Glu Leu Thr Tyr Gly Ile Lys 20 25
30Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn
35 40 45Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Cys Val
Ser 50 55 60Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys
Glu Cys65 70 75 80Phe Thr Thr6683PRTArtificialSynthetic construct
66Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe1
5 10 15Lys Pro Leu Ala Glu Ile Asp Gly Cys Glu Leu Thr Tyr Gly Ile
Lys 20 25 30Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp
Glu Asn 35 40 45Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr
Glu Val Ser 50 55 60Leu Ile Cys Arg Arg Gly Asp Met Ser Ser Asn Pro
Ala Lys Glu Thr65 70 75 80Phe Thr Thr6783PRTArtificialSynthetic
construct 67Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr
Trp Phe1 5 10 15Lys Pro Leu Ala Glu Ile Asp Gly Cys Glu Leu Thr Tyr
Gly Ile Lys 20 25 30Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu Thr
Glu Asp Glu Asn 35 40 45Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr
Glu Tyr Cys Val Ser 50 55 60Leu Ile Cys Arg Arg Gly Asp Met Ser Ser
Asn Pro Ala Lys Glu Cys65 70 75 80Phe Thr
Thr6891PRTArtificialSynthetic construct 68Pro Ala Ile Ser Asn Val
Arg Val Ser Asp Val Thr Asn Ser Ser Ala1 5 10 15Thr Ile Arg Trp Asp
Val Ser Leu Ala Ala Asn Asn Arg Val Leu Phe 20 25 30Ser Thr Asn Ser
Asp Leu Ser Ser Pro Gln Trp Ser Ala Trp Asp Asn 35 40 45Ser Thr Asp
Ser Pro Met Ile Thr Leu Ser Gly Leu Ser Ala Gly Thr 50 55 60Ala Tyr
Tyr Phe Ser Val Tyr Ser Phe Arg Pro Asp Asn Ala Ser Leu65 70 75
80Tyr Ser Asn Ser Ser Ile Met Ser Phe Thr Thr 85
906989PRTArtificialSynthetic construct 69Pro Pro Pro Lys Pro Val
Ile Arg Phe Ala Gln Ala Gly Asn Asn Ser1 5 10 15Ile Ser Leu Ser Trp
Tyr Asp Thr Asn Thr Ser Gly Tyr Tyr Ile Gln 20 25 30Trp Trp Ser Ser
Ile Asp Asn Asn Lys Ser Thr Ile Asn Val Gly Asn 35 40 45Val Ser Ser
Tyr Leu Phe Ile Asn Leu Thr Asn Gly Val Thr Tyr Tyr 50 55 60Phe Arg
Ile Ile Pro Tyr Asn Gln Ala Gly Asn Gly Thr Ser Ser Asp65 70 75
80Ile Ile Ser Leu Thr Pro Gly Ala Val 857089PRTArtificialSynthetic
construct 70Pro Asp Ser Pro Ser Val Lys Val Ile Val Gly Asp Arg Asn
Ala Thr1 5 10 15Val Ile Trp Ser Lys Pro Tyr Asn Gly Gly Phe Pro Ile
Leu Gly Tyr 20 25 30Tyr Leu Thr Val Lys Thr Asp Asn Ser Ser Tyr Thr
Ile Asn Val Gly 35 40 45Asn Val Ser Lys Tyr Thr Leu Thr Asn Leu Thr
Pro Glu Val Leu Tyr 50 55 60Glu Val Met Val Val Ala Tyr Asn Lys Leu
Gly Asn Ser Ser Pro Gly65 70 75 80Ile Val Asn Phe Val Ala Leu Thr
Thr 857187PRTArtificialSynthetic construct 71Leu Thr Thr Ala Ser
Ile Ser Val Ser Val Tyr Lys Lys Val Asn Gly1 5 10 15Val Leu Ile Ser
Trp Asn Lys Thr Glu Asn Thr Thr Tyr Asn Leu Leu 20 25 30Ile Ser Asp
Lys Lys Gly Lys Ile Ile Val Asn Ile Thr Thr Thr Asn 35 40 45Thr Ser
Tyr Phe Ala Tyr Ile Pro Tyr Gly Ile Tyr Asn Val Thr Ile 50 55 60Arg
Ala Thr Asn Gln Val Gly Thr Asn Ser Thr Ser Phe Pro Ile Val65 70 75
80Phe Tyr Ile Pro Pro Phe Ile 857284PRTArtificialSynthetic
construct 72Pro Leu Val Lys Phe Ser Ile Gly Asn Asn Ser Ile Leu Asn
Leu Lys1 5 10 15Trp Asn Asn Val Thr Gly Ala Thr Phe Tyr Leu Val Tyr
Val Asn Thr 20 25 30Thr Leu Ile Ala Asn Val Thr Thr Asp Ser Tyr Ser
Leu Asn Leu Thr 35 40 45Pro Gly Phe His Val Ile Arg Val Val Ala Ala
Asn Pro Ile Tyr Asn 50 55 60Ser Ser Pro Ala Ser Leu Gly Ile Leu Ile
Gln Gln His Ser Val Thr65 70 75 80Ser Ser Ile
Thr7388PRTArtificialSynthetic construct 73Pro Leu Pro Pro Lys Ile
Thr Ser Tyr Ser Ala Gly Asn Glu Ser Val1 5 10 15Thr Leu Gly Trp Asn
Pro Val Arg Leu Ser Ser Gly Tyr Glu Ile Ile 20 25 30Tyr Trp Asn Asn
Met Gly Phe Asn Ser Ser Ile Asn Val Gly Asn Val 35 40 45Thr Ser Tyr
Thr Val Thr Gly Leu Lys Asp Gly Ile Thr Tyr Tyr Phe 50 55 60Glu Val
Leu Ala Tyr Asn Ser Ile Gly Tyr Ser Ser Pro Ser Ser Ile65 70 75
80Ile Ala Leu Thr Pro Ala Ser Val 857492PRTArtificialSynthetic
construct 74Pro Asn Pro Pro Gln Leu Val Ser Val Lys Tyr Gly Asn Asp
Asn Val1 5 10 15Thr Leu Asn Trp Leu Pro Pro Thr Phe Ser Gly Gly Tyr
Leu Leu Leu 20 25 30Gly Tyr Tyr Val Ile Val Lys Asn Glu Asn Ser Met
Val Ser Ser His 35 40 45Phe Val Asn Ser Thr Ser Leu Thr Ile Ser Asn
Leu Thr Pro Asn Val 50 55 60Thr Tyr Asn Val Phe Ile Tyr Ala Val Asn
Lys Leu Gly Asn Ser Ser65 70 75 80Pro Leu Val Leu Thr Val Val Pro
Ile Thr Lys Ala 85 907586PRTArtificialSynthetic construct 75Pro Ile
Thr Lys Ala Ser Val Phe Ala Phe Ile Thr Lys Leu Gly Asn1 5 10 15Gly
Ile Leu Val Asn Trp Thr Thr Ser Phe Pro Ala Asn Ile Thr Leu 20 25
30Glu Leu Tyr Asn Pro Asn Gly Asn Leu Ile Ser Gln Ile Ala Ala Ile
35 40 45Lys Gly Asn Ser Ser Tyr Leu Phe Arg Val Pro Gln Gly Asn Tyr
Thr 50 55 60Leu Val Ile Ile Ala Ser Asn Ser Ala Gly Val Ser Lys Tyr
Val Tyr65 70 75 80Gln Val Val Tyr Tyr Leu
857683PRTArtificialSynthetic construct 76Pro Pro Ala Ser Pro Gln
Val Ser Leu Ile Gly Phe Gly Asn Asn Leu1 5 10 15Tyr Ile Ser Trp Asn
Asn Glu Ala Asn Val Ile Thr Tyr Leu Val Tyr 20 25 30Val Asn Asn Ser
Leu Val Tyr Glu Gly Pro Ser Asn Ser Ile Val Thr 35 40 45Asn Ile Ser
Asn Gly Thr Tyr Leu Val Lys Val Ile Gly Val Asn Pro 50 55 60Ala Gly
Ser Ser Ser Pro Gly Ile Ala Val Ile His Tyr Thr Gly Asp65 70 75
80Tyr Val Thr7783PRTArtificialSynthetic construct 77Ala Ser Lys Ala
Asn Leu Thr Val Thr Val Tyr Lys Lys Ile Asn Gly1 5 10 15Phe Leu Val
Ser Trp Asn Ser Thr Ser Lys Ala Lys Tyr Ile Leu Thr 20 25 30Val Ser
Lys Glu Asn Val Val Leu Leu Asn Val Ser Thr Thr Asn Thr 35 40 45Ser
Tyr Phe Val Lys Val Pro Phe Gly Val Tyr Asn Ile Ser Leu Glu 50 55
60Ala Val Asn Ile Val Gly Ile Thr Lys Tyr Ala Phe Ile Leu Ile Tyr65
70 75 80Tyr Ile Gln7881PRTArtificialSynthetic construct 78Pro Ala
Ser Pro Thr Val Asn Trp Ser Ile Thr Leu Asn Thr Val Ser1 5 10 15Leu
Asn Trp Ser Lys Val Ser Gly Ala Glu Tyr Tyr Leu Ile Tyr Asp 20 25
30Asn Gly Lys Leu Ile Thr Asn Thr Thr Asn Thr Ala Phe Thr Phe Asn
35 40 45Leu Thr Ile Gly Gln Asn Glu Ile Glu Val Tyr Ala Ala Asn Ala
Tyr 50 55 60Tyr Lys Ser Ala Pro Tyr Ile Ile Asn Asp Val Arg Asn Tyr
Ile Val65 70 75 80Val79105PRTArtificialSynthetic construct 79Ala
Ala Pro Ala Ile Ser Asn Val Arg Val Ser Asp Val Thr Asn Ser1 5 10
15Ser Ala Thr Ile Arg Trp Asp Val Ser Leu Ala Ala Asn Asn Arg Val
20 25 30Leu Phe Ser Thr Asn Ser Asp Leu Ser Ser Pro Gln Trp Ser Ala
Trp 35 40 45Asp Asn Ser Thr Asp Ser Pro Met Ile Thr Leu Ser Gly Leu
Ser Ala 50 55 60Gly Thr Ala Tyr Tyr Phe Ser Val Tyr Ser Phe Arg Pro
Asp Asn Ala65 70 75 80Ser Leu Tyr Ser Asn Ser Ser Ile Met Ser Phe
Thr Thr Gly Gly Gly 85 90 95Thr Leu Glu His His His His His His 100
1058091PRTArtificialSynthetic construct 80Pro Ala Ile Ser Asn Val
Arg Val Ser Asp Val Thr Asn Ser Ser Ala1 5 10 15Thr Ile Arg Trp Asp
Val Ser Leu Ala Ala Asn Asn Arg Val Leu Phe 20 25 30Ser Thr Asn Ser
Asp Leu Ser Ser Pro Gln Trp Ser Ala Trp Asp Asn 35 40 45Ser Thr Asp
Ser Pro Met Ile Thr Leu Ser Gly Leu Ser Ala Gly Thr 50 55 60Ala Tyr
Tyr Phe Ser Val Tyr Ser Phe Arg Pro Asp Asn Ala Ser Leu65 70 75
80Tyr Ser Asn Ser Ser Ile Met Ser Phe Thr Thr 85
9081108PRTArtificialSynthetic construct 81Ala Ala Ser Glu Pro Gln
Asn Leu Lys Ala Thr Ala Gly Asn Asn Asn1 5 10 15Ile Thr Leu Thr Trp
Asp Pro Pro Ile Asp Asp Gly Gly Cys Arg Ile 20 25 30Val Glu Tyr Arg
Ile Tyr Arg Gly Thr Asn Asn Asn Asn Leu Glu Tyr 35 40 45Tyr Ala Ser
Val Asn Gly Ser Thr Thr Thr Phe Ile Asp Lys Asn Ile 50 55 60Val Tyr
Ser Gln Thr Tyr Tyr Tyr Lys Val Ser Ala Val Asn Asn Ile65 70 75
80Val Glu Gly Pro Lys Ser Asn Thr Ala Ser Ala Thr Pro Thr Ser Ser
85 90 95Gly Gly Gly Thr Leu Glu His His His His His His 100
1058294PRTArtificialSynthetic construct 82Ser Glu Pro Gln Asn Leu
Lys Ala Thr Ala Gly Asn Asn Asn Ile Thr1 5 10 15Leu Thr Trp Asp Pro
Pro Ile Asp Asp Gly Gly Cys Arg Ile Val Glu 20 25 30Tyr Arg Ile Tyr
Arg Gly Thr Asn Asn Asn Asn Leu Glu Tyr Tyr Ala 35 40 45Ser Val Asn
Gly Ser Thr Thr Thr Phe Ile Asp Lys Asn Ile Val Tyr 50 55 60Ser Gln
Thr Tyr Tyr Tyr Lys Val Ser Ala Val Asn Asn Ile Val Glu65 70 75
80Gly Pro Lys Ser Asn Thr Ala Ser Ala Thr Pro Thr Ser Ser 85
9083102PRTArtificialSynthetic construct 83Ala Ala Pro Leu Pro Pro
Lys Ile Thr Ser Tyr Ser Ala Gly Asn Glu1 5 10 15Ser Val Thr Leu Gly
Trp Asn Pro Val Arg Leu Ser Ser Gly Tyr Glu 20 25 30Ile Ile Tyr Trp
Asn Asn Met Gly Phe Asn Ser Ser Ile Asn Val Gly 35 40 45Asn Val Thr
Ser Tyr Thr Val Thr Gly Leu Lys Asp Gly Ile Thr Tyr 50 55 60Tyr Phe
Glu Val Leu Ala Tyr Asn Ser Ile Gly Tyr Ser Ser Pro Ser65 70 75
80Ser Ile Ile Ala Leu Thr Pro Ala Ser Val Gly Gly Gly Thr Leu Glu
85 90 95His His His His His His 1008488PRTArtificialSynthetic
cosntruct 84Pro Leu Pro Pro Lys Ile Thr Ser Tyr Ser Ala Gly Asn Glu
Ser Val1 5 10 15Thr Leu Gly Trp Asn Pro Val Arg Leu Ser Ser Gly Tyr
Glu Ile Ile 20 25 30Tyr Trp Asn Asn Met Gly Phe Asn Ser Ser Ile Asn
Val Gly Asn Val 35 40 45Thr Ser Tyr Thr Val Thr Gly Leu Lys Asp Gly
Ile Thr Tyr Tyr Phe 50 55 60Glu Val Leu Ala Tyr Asn Ser Ile Gly Tyr
Ser Ser Pro Ser Ser Ile65 70 75 80Ile Ala Leu Thr Pro Ala Ser Val
8585101PRTArtificialSynthetic construct 85Ala Ala Pro Pro Lys Pro
Gln Ile Ala Ser Ile Ala Ser Gly Asn Glu1 5 10 15Thr Ile Thr Val Lys
Trp Tyr Asp Thr Asn Ala Ser Gly Tyr Tyr Ile 20 25 30Thr Tyr Trp Ser
Asn Phe Ser Gln Lys Val Thr Ile Asn Val Gly Asn 35 40 45Val Thr Ser
Tyr Thr Ile Lys His Leu Lys Asp Gly Val Thr Tyr Tyr 50 55 60Ile Gln
Ile Val Pro Tyr Asn Ser Leu Gly Asn Gly Thr Pro Ser Asp65 70 75
80Ile Ile Ser Ala Thr Pro Ser Ser Val Gly Gly Gly Thr Leu Glu His
85 90 95His His His His His 1008687PRTArtificialSynthetic construct
86Pro Pro Lys Pro Gln Ile Ala Ser Ile Ala Ser Gly Asn Glu Thr Ile1
5 10 15Thr Val Lys Trp Tyr Asp Thr Asn Ala Ser Gly Tyr Tyr Ile Thr
Tyr 20 25 30Trp Ser Asn Phe Ser Gln Lys Val Thr Ile Asn Val Gly Asn
Val Thr 35 40 45Ser Tyr Thr Ile Lys His Leu Lys Asp Gly Val Thr Tyr
Tyr Ile Gln 50 55 60Ile Val Pro Tyr Asn Ser Leu Gly Asn Gly Thr Pro
Ser Asp Ile Ile65 70 75 80Ser Ala Thr Pro Ser Ser Val
8587100PRTArtificialSynthetic construct 87Ala Ala Pro Asn Pro Pro
Ile Ile Lys Val Lys Ile Gly Asn Leu Asn1 5 10 15Ala Thr Leu Thr Trp
Tyr Asp Thr Phe Asn Gly Gly Tyr Pro Ile Glu 20 25 30Gly Tyr Tyr Leu
Tyr Val Asn Gly Lys Gly Ile Asn Val Gly Asn Ile
35 40 45Thr Ser Tyr Val Leu Thr Asn Leu Thr Ala Gly Glu Leu Tyr Thr
Ile 50 55 60Glu Leu Ile Ala Tyr Asn Lys Ile Gly Asn Ser Ser Ile Ser
Ser Val65 70 75 80Ser Phe Ile Ala Ala Ser Lys Ala Gly Gly Gly Thr
Leu Glu His His 85 90 95His His His His
1008886PRTArtificialSynthetic construct 88Pro Asn Pro Pro Ile Ile
Lys Val Lys Ile Gly Asn Leu Asn Ala Thr1 5 10 15Leu Thr Trp Tyr Asp
Thr Phe Asn Gly Gly Tyr Pro Ile Glu Gly Tyr 20 25 30Tyr Leu Tyr Val
Asn Gly Lys Gly Ile Asn Val Gly Asn Ile Thr Ser 35 40 45Tyr Val Leu
Thr Asn Leu Thr Ala Gly Glu Leu Tyr Thr Ile Glu Leu 50 55 60Ile Ala
Tyr Asn Lys Ile Gly Asn Ser Ser Ile Ser Ser Val Ser Phe65 70 75
80Ile Ala Ala Ser Lys Ala 8589297DNAArtificialSynthetic construct
89gccatggcag ccccggcgat tagcaatgtg cgcgttagcg atgtgaccaa cagcagcgcc
60accattcgtt gggatgtgag cctggcggcg aataatcgcg tgctgtttag caccaacagc
120gatctgagca gcccgcagtg gagcgcgtgg gataacagca ccgatagccc
gatgattacc 180ctgagcggtc tgagcgcggg caccgcgtat tattttagcg
tgtatagctt tcgtccggat 240aatgcgagcc tgtatagcaa cagcagcatt
atgagcttta ccaccggtgg cggtacc 29790273DNAArtificialSynthetic
construct 90ccggcgatta gcaatgtgcg cgttagcgat gtgaccaaca gcagcgccac
cattcgttgg 60gatgtgagcc tggcggcgaa taatcgcgtg ctgtttagca ccaacagcga
tctgagcagc 120ccgcagtgga gcgcgtggga taacagcacc gatagcccga
tgattaccct gagcggtctg 180agcgcgggca ccgcgtatta ttttagcgtg
tatagctttc gtccggataa tgcgagcctg 240tatagcaaca gcagcattat
gagctttacc acc 27391306DNAArtificialSynthetic construct
91gccatggccg ccagcgaacc gcagaacctg aaagcgaccg cgggtaataa caatattacc
60ctgacctggg atccgccgat tgatgatggt ggctgccgca ttgtggaata tcgtatttat
120cgtggcacca ataataacaa cctggaatat tatgcgagcg ttaacggcag
caccaccacc 180tttattgata aaaatattgt gtatagccag acctattatt
ataaagtgag cgcggtgaac 240aatattgtgg aaggcccgaa aagcaacacc
gcgagcgcga ccccgaccag cagcggtggc 300ggtacc
30692282DNAArtificialSynthetic cosntruct 92agcgaaccgc agaacctgaa
agcgaccgcg ggtaataaca atattaccct gacctgggat 60ccgccgattg atgatggtgg
ctgccgcatt gtggaatatc gtatttatcg tggcaccaat 120aataacaacc
tggaatatta tgcgagcgtt aacggcagca ccaccacctt tattgataaa
180aatattgtgt atagccagac ctattattat aaagtgagcg cggtgaacaa
tattgtggaa 240ggcccgaaaa gcaacaccgc gagcgcgacc ccgaccagca gc
28293288DNAArtificialSynthetic cosntruct 93gccatggccg ccccgctccc
accgaaaatt accagctata gcgcgggcaa cgaaagcgtg 60accctgggct ggaacccggt
gcgtctgagc agcggctatg aaattattta ttggaacaat 120atgggcttta
acagcagcat taatgtgggt aatgtgacca gctataccgt gaccggcctg
180aaagatggca ttacctatta ttttgaagtg ctggcctata acagcattgg
ttatagcagc 240ccgagcagca ttatcgcgct gaccccggcg agcgtgggtg gcggtacc
28894264DNAArtificialSynthetic construct 94ccgctcccac cgaaaattac
cagctatagc gcgggcaacg aaagcgtgac cctgggctgg 60aacccggtgc gtctgagcag
cggctatgaa attatttatt ggaacaatat gggctttaac 120agcagcatta
atgtgggtaa tgtgaccagc tataccgtga ccggcctgaa agatggcatt
180acctattatt ttgaagtgct ggcctataac agcattggtt atagcagccc
gagcagcatt 240atcgcgctga ccccggcgag cgtg
26495285DNAArtificialSynthetic construct 95gccatggccg ccccgccgaa
accgcagatt gccagcattg ccagcggtaa tgaaaccatt 60accgtgaaat ggtatgatac
caatgcgagc ggctattata ttacctattg gagcaatttt 120agccagaaag
tgaccattaa tgtgggtaac gtgaccagct ataccattaa acatctgaaa
180gatggcgtga cctattatat tcagattgtg ccgtataaca gcctgggcaa
tggcaccccg 240agcgatatta ttagcgcgac cccgagcagc gttggtggcg gtacc
28596261DNAArtificialSynthetic construct 96ccgccgaaac cgcagattgc
cagcattgcc agcggtaatg aaaccattac cgtgaaatgg 60tatgatacca atgcgagcgg
ctattatatt acctattgga gcaattttag ccagaaagtg 120accattaatg
tgggtaacgt gaccagctat accattaaac atctgaaaga tggcgtgacc
180tattatattc agattgtgcc gtataacagc ctgggcaatg gcaccccgag
cgatattatt 240agcgcgaccc cgagcagcgt t
26197282DNAArtificialSynthetic construct 97gccatggccg ccccgaatcc
gccgattatt aaagtgaaaa ttggcaatct gaatgcgacc 60ctgacctggt atgatacctt
taatggtggt tatccgattg aaggctatta tctgtatgtg 120aacggtaaag
gtattaacgt gggcaacatt accagctatg tgctgaccaa tctgaccgcc
180ggtgaactgt ataccattga actgattgcg tataacaaaa tcggcaacag
cagcattagc 240agcgtgagct ttattgcggc gagcaaagcg ggtggcggta cc
28298258DNAArtificialSynthetic construct 98ccgaatccgc cgattattaa
agtgaaaatt ggcaatctga atgcgaccct gacctggtat 60gataccttta atggtggtta
tccgattgaa ggctattatc tgtatgtgaa cggtaaaggt 120attaacgtgg
gcaacattac cagctatgtg ctgaccaatc tgaccgccgg tgaactgtat
180accattgaac tgattgcgta taacaaaatc ggcaacagca gcattagcag
cgtgagcttt 240attgcggcga gcaaagcg 258993994DNAArtificialSynthetic
construct 99gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
ataatggttt 60cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
tgtttatttt 120tctaaataca ttcaaatatg tatccgctca tgagacaata
accctgataa atgcttcaat 180aatattgaaa aaggaagagt atgagtattc
aacatttccg tgtcgccctt attccctttt 240ttgcggcatt ttgccttcct
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300ctgaagatca
gttgggtgct cgagtgggtt acatcgaact ggatctcaac agcggtaaga
360tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
aaagttctgc 420tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
gcaactcggt cgccgcatac 480actattctca gaatgacttg gttgagtact
caccagtcac agaaaagcat cttacggatg 540gcatgacagt aagagaatta
tgcagtgctg ccataaccat gagtgataac actgcggcca 600acttacttct
gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
660gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
ataccaaacg 720acgagcgtga caccacgatg cctgtagcaa tggcaacaac
gttgcgcaaa ctattaactg 780gcgaactact tactctagct tcccggcaac
aattaataga ctggatggag gcggataaag 840ttgcaggacc acttctgcgc
tcggcccttc cggctggctg gtttattgct gataaatctg 900gagccggtga
gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
960cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
cgaaatagac 1020agatcgctga gataggtgcc tcactgatta agcattggta
actgtcagac caagtttact 1080catatatact ttagattgat ttaaaacttc
atttttaatt taaaaggatc taggtgaaga 1140tcctttttga taatctcatg
accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200cagaccccgt
agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
1260gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
gatcaagagc 1320taccaactct ttttccgaag gtaactggct tcagcagagc
gcagatacca aatactgtcc 1380ttctagtgta gccgtagtta ggccaccact
tcaagaactc tgtagcaccg cctacatacc 1440tcgctctgct aatcctgtta
ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500ggttggactc
aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
1560cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac
ctacagcgtg 1620agctatgaga aagcgccacg cttcccgaag ggagaaaggc
ggacaggtat ccggtaagcg 1680gcagggtcgg aacaggagag cgcacgaggg
agcttccagg gggaaacgcc tggtatcttt 1740atagtcctgt cgggtttcgc
cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800gggggcggag
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1860gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
gataaccgta 1920ttaccgcctt tgagtgagct gataccgctc gccgcagccg
aacgaccgag cgcagcgagt 1980cagtgagcga ggaagcggaa gagcgcccaa
tacgcaaacc gcctctcccc gcgcgttggc 2040cgattcatta atgcagctgg
cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100acgcaattaa
tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
2160cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
aacagctatg 2220accatgatta cgccaagctt tggagccttt tttttggaga
ttttcaacgt gaaaaaatta 2280ttattcgcaa ttcctttagt tgttcctttc
tatgcggccc agccggccat ggccgccatt 2340gaagtgaaag atgtgaccga
taccaccgcg ctgattacct ggtttaaacc gctggcggaa 2400attgatggct
gtgaactgac ctatggcatt aaagatgtgc cgggcgatcg caccaccata
2460gatctgaccg aagatgaaaa ccagtatagc attggtaacc tgaaaccgga
taccgaatat 2520gaagtgagcc tgatttgccg tcgtggcgat atgagcggcg
cgccggcgaa agaaaccttt 2580accaccggtg gcggtacccc aaccgacccg
ccaaccactc caccaactga tagcccaggc 2640ggtactggtg gctctggttc
cggtgatttt gattatgaaa agatggcaaa cgctaataag 2700ggggctatga
ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa aggcaaactt
2760gattctgtcg ctactgatta cggtgctgct atcgatggtt tcattggtga
cgtttccggc 2820cttgctaatg gtaatggtgc tactggtgat tttgctggct
ctaattccca aatggctcaa 2880gtcggtgacg gtgataattc acctttaatg
aataatttcc gtcaatattt accttccctc 2940cctcaatcgg ttgaatgtcg
cccttttgtc tttagcgctg gtaaaccata tgaattttct 3000attgattgtg
acaaaataaa cttattccgt ggtgtctttg cgtttctttt atatgttgcc
3060acctttatgt atgtattttc tacgtttgct aacatactgc gtaataagga
gtcttaagaa 3120ttcgacggtc acagcttgtc tgtaagcgga tgccgggagc
agacaagccc gtcagggcgc 3180gtcagcgggt gttggcgggt gtcggggctg
gcttaactat gcggcatcag agcagattgt 3240actgagactg caccataaaa
ttgtaaacgt taatattttg ttaaaattcg cgttaaattt 3300ttgttaaatc
agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
3360aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga
gtccactatt 3420aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
tatcagggcg atggcccact 3480acgtgaacca tcacccaaat caagtttttt
ggggtcgagg tgccgtaaag cactaaatcg 3540gaaccctaaa gggagccccc
gatttagagc ttgacgggga aagccggcga acgtggcgag 3600aaaggaaggg
aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
3660gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
cgtactatgg 3720ttgctttgac gtatgcggtg tgaaataccg cacagatgcg
taaggagaaa ataccgcatc 3780aggcgccatt cgccattcag gctgcgcaac
tgttgggaag ggcgatcggt gcgggcctct 3840tcgctattac gccagctggc
gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 3900ccagggtttt
cccagtcacg acgttgtaaa acgacggcca gttccgggag ctgcatgtgt
3960cagaggtttt caccgtcatc accgaaacgc gcga
399410011PRTArtificialSynthetic construct 100Ser Pro Pro Ser Val
Leu Val Gly Tyr Thr Gly1 5 101019PRTArtificialSynthetic construct
101Val Thr Glu Phe Gly Arg Arg Arg Ser1 51029PRTArtificialSynthetic
cosntruct 102Phe Lys Pro Leu Ala Glu Ile Asp Gly1
510311PRTArtificialSynthetic construct 103Arg Lys Ile Ile Gly Leu
Leu Ser Asn Pro Ala1 5 1010411PRTArtificialSynthetic construct
104Gly Thr Val Val Gly Gln Lys Ser Asn Pro Ala1 5
1010511PRTArtificialSynthetic construct 105Ser Pro Gly Glu Arg Ile
Trp Met Phe Thr Gly1 5 1010610PRTArtificialSynthetic construct
106Pro Asn Tyr Glu Arg Ile Ser Asn Pro Ala1 5
1010711PRTArtificialSynthetic construct 107Ser Pro Ser Gly Arg Val
Ile Leu Trp Thr Gly1 5 1010811PRTArtificialSynthetic construct
108Asp Asn Leu Tyr Gly Arg Ile Ser Asn Pro Ala1 5
1010912PRTArtificialSynthetic construct 109Ala Thr Pro Gly Cys Arg
Asn Gly Lys Ile Val Gly1 5 1011011PRTArtificialSynthetic construct
110Thr Thr Ser Val Gly Ala Thr Ser Asn Pro Ala1 5
101119PRTArtificialSynthetic construct 111Phe Lys Pro Leu Ala Glu
Ile Asp Gly1 51129PRTArtificialSynthetic construct 112Tyr Asn Arg
Tyr Gly Leu Cys Pro Ser1 51139PRTArtificialSynthetic construct
113Ser Asn Arg Ile Gly Met Cys Pro Ser1
511412PRTArtificialSynthetic construct 114Ala Gln Pro Thr Ser Pro
Asn Gly Ser Ile Xaa Gly1 5 1011510PRTArtificialSynthetic construct
115Arg Arg Gly Asp Met Ser Ser Asn Pro Ala1 5
1011611PRTArtificialSynthetic construct 116Asp Thr Val His Gly Arg
Leu Ser Asn Pro Ala1 5 1011711PRTArtificialSynthetic construct
117Arg Lys Val Leu Gly Arg Leu Ser Asn Pro Ala1 5
101189PRTArtificialSynthetic construct 118Arg Lys Leu Val Gly Ala
Leu Arg Ser1 511910PRTArtificialSynthetic construct 119Arg Lys Val
Leu Arg Tyr Ser Asn Pro Ala1 5 1012011PRTArtificialSynthetic
construct 120Ser Pro Cys Asn Gly Gly Lys Arg Cys Thr Gly1 5
1012110PRTArtificialSynthetic construct 121Arg Arg Gly Asp Met Ser
Ser Asn Pro Ala1 5 101229PRTArtificialSynthetic construct 122Phe
Lys Trp Leu Gly Ala Ile Arg Ser1 51239PRTArtificialSynthetic
construct 123Gly Asn Cys Val Gly Asn Leu Trp Ser1
512411PRTArtificialSynthetic construct 124Ser Pro Ala Trp Ile Thr
Trp His Arg Thr Gly1 5 101259PRTArtificialSynthetic construct
125His Thr Pro Leu Gly His Leu Arg Ser1
512611PRTArtificialSynthetic construct 126Ser Pro Cys Ile Met Val
Cys Leu Arg Thr Gly1 5 1012710PRTArtificialSynthetic construct
127Arg Arg Gly Asp Met Ser Gly Ala Pro Ala1 5
1012811PRTArtificialSynthetic construct 128Ser Pro Cys Leu Phe Val
Cys Leu Arg Thr Gly1 5 1012910PRTArtificialSynthetic construct
129Arg Arg Gly Asp Met Ser Gly Ala Pro Ala1 5
1013011PRTArtificialSynthetic construct 130Ser Pro Pro Leu Phe Cys
Cys Gln Lys Thr Gly1 5 101319PRTArtificialSynthetic construct
131Phe Lys Leu Thr Gly Phe Leu Tyr Ser1
513211PRTArtificialSynthetic construct 132Ser Pro Ser Val Ala Arg
Met Leu Glu Thr Gly1 5 101339PRTArtificialSynthetic construct
133Ile Thr Leu Cys Gly Arg Gly Val Ser1
513411PRTArtificialSynthetic construct 134Ser Pro Pro Glu Tyr Ala
Phe Tyr Tyr Thr Gly1 5 1013511PRTArtificialSynthetic construct
135Val Lys Asn Cys Gly Leu Phe Ser Asn Pro Ala1 5
101369PRTArtificialSynthetic construct 136Ser Leu Ala Pro Gly Tyr
Arg Leu Gly1 513710PRTArtificialSynthetic construct 137Val Lys Leu
Cys Met Arg Gly Asn Pro Ala1 5 1013812PRTArtificialSynthetic
construct 138Ala Thr Pro Ser Val Phe Asp Ser His Ile Glu Gly1 5
101399PRTArtificialSynthetic construct 139Trp Lys His His Gly Asp
Ala Trp Ser1 514012PRTArtificialSynthetic construct 140Ala Lys Pro
Ser Ile Val Asn Gly Phe Ile Ser Gly1 5 101419PRTArtificialSynthetic
construct 141Asp Lys Cys Phe Gly Ala Met Lys Ser1
514212PRTArtificialSynthetic construct 142Ala Lys Pro Met Ser Cys
Ser Gly Tyr Ile Gln Gly1 5 101439PRTArtificialSynthetic construct
143Ala Lys Leu Thr Gly Trp Leu Cys Ser1
514412PRTArtificialSynthetic construct 144Ala Ala Pro Phe Phe Gly
Ser Ser Tyr Ile Ser Gly1 5 101456PRTArtificialSynthetic construct
145His Tyr Tyr Val Thr Arg1 51469PRTArtificialSynthetic construct
146Val Asn Leu Ser Gly His Met Pro Ser1
514712PRTArtificialSynthetic construct 147Ala Pro Pro Met Leu Thr
Asp Ser Glu Ile Asn Gly1 5 101486PRTArtificialSynthetic construct
148Thr Ser Ser Tyr Trp Ser1 51499PRTArtificialSynthetic construct
149Ser Thr Leu Arg Arg Asn Ala Ile Ser1
515012PRTArtificialSynthetic construct 150Ala Lys Pro Glu Lys Trp
Asp Gly Ser Ile Tyr Gly1 5 101516PRTArtificialSynthetic construct
151Asn Ser Arg His Thr Ala1 515211PRTArtificialSynthetic construct
152Phe Thr Pro Tyr Gly Ala Lys Ser Asn Pro Ala1 5
1015312PRTArtificialSynthetic construct 153Ala Pro Pro Pro Phe Ser
Asn Ser Cys Ile Ile Gly1 5 101546PRTArtificialSynthetic construct
154Arg Pro Gly Arg Ala Ser1 515511PRTArtificialSynthetic construct
155Ser Thr Gly Thr Gly Leu Pro Ser Asn Pro Ala1 5
1015611PRTArtificialSynthetic construct 156Ser Pro Cys Cys Pro Tyr
Asp Arg Tyr Thr Gly1 5 101576PRTArtificialSynthetic construct
157Gln Ser Ser Arg Ser His1 515811PRTArtificialSynthetic construct
158Ile Thr Thr Phe Gly His Val Ser Asn Pro Ala1 5
1015912PRTArtificialSynthetic construct 159Ala Lys Pro Arg Gln Gly
Gly Ser Asn Ile Ser Gly1 5 101606PRTArtificialSynthetic construct
160Tyr His Lys Gly Leu His1 51619PRTArtificialSynthetic construct
161Pro Lys Met Thr Gly Tyr Thr Tyr Ser1
516211PRTArtificialSynthetic construct 162Ser Pro Gly Pro Leu Leu
Arg His Thr Thr Gly1 5 101636PRTArtificialSynthetic construct
163Arg Pro Ile Pro Arg Ala1 516410PRTArtificialSynthetic construct
164Arg Asn Arg Pro Gln Gln Ser Asn Pro Ala1 5
1016511PRTArtificialSynthetic construct 165Ser Pro Gly Gly Phe Gln
Lys
Ile Thr Thr Gly1 5 101666PRTArtificialSynthetic construct 166Val
Asn Arg Arg Asn His1 51679PRTArtificialSynthetic construct 167Leu
Thr Tyr Lys Ala Arg Ala Ile Ser1 516811PRTArtificialSynthetic
construct 168Ser Pro Arg Met Tyr Thr Trp Ile Gln Thr Gly1 5
101696PRTArtificialSynthetic construct 169Thr His Leu Ser Gly Ser1
51709PRTArtificialSynthetic construct 170Leu Lys Leu Thr Arg Thr
His Ile Ser1 51719PRTArtificialSynthetic construct 171Ser His Ala
Gly Gly Ile Arg Ile Gly1 51726PRTArtificialSynthetic construct
172His Val Trp Gln Val Tyr1 517310PRTArtificialSynthetic construct
173Met Thr Pro Tyr Leu Leu Gly Asn Pro Ala1 5
1017411PRTArtificialSynthetic construct 174Ser Pro Ser His Gly Val
Glu Ser Ser Thr Gly1 5 101756PRTArtificialSynthetic construct
175His Gly Leu Gln Arg Val1 51769PRTArtificialSynthetic construct
176Ala Lys Ile Cys Gly His Leu Val Ser1
517711PRTArtificialSynthetic construct 177Ser Pro Cys Gln Leu Leu
Ala Leu Ile Thr Gly1 5 101786PRTArtificialSynthetic construct
178Asn Ser Arg His Tyr His1 517911PRTArtificialSynthetic construct
179Tyr Thr Ser Thr Gly Gln Arg Ser Asn Pro Ala1 5
1018011PRTArtificialSynthetic construct 180Ser Pro Cys Gln Met Leu
Ser Ser Leu Thr Gly1 5 101816PRTArtificialSynthetic construct
181Asn Ile Glu Arg Pro Lys1 518211PRTArtificialSynthetic construct
182Phe Thr Met Thr Gly Tyr Arg Ser Asn Pro Ala1 5
1018311PRTArtificialSynthetic construct 183Ser Pro Cys Cys Gln Glu
Phe Thr Leu Thr Gly1 5 101846PRTArtificialSynthetic construct
184His Asn His His His His1 518511PRTArtificialSynthetic construct
185Ile Thr Asp Ala Gly Asn Lys Ser Asn Pro Ala1 5
1018611PRTArtificialSynthetic construct 186Ser Pro Cys Ser Pro Cys
Gln Leu Val Thr Gly1 5 101876PRTArtificialSynthetic construct
187Ser Cys Thr Arg Ala Lys1 518811PRTArtificialSynthetic construct
188Ile Asn Lys Leu Gly Asp Thr Ser Asn Pro Ala1 5
1018911PRTArtificialSynthetic construct 189Ser Pro Ser Arg Gly Gly
Thr Ser Leu Thr Gly1 5 101906PRTArtificialSynthetic construct
190Asp Gln Val Arg Ala Thr1 519111PRTArtificialSynthetic construct
191His Thr Asn Ser Gly Gln Pro Ser Asn Pro Ala1 5
1019211PRTArtificialSynthetic construct 192Ser Pro Gly Met Phe Asp
Gln Val Arg Thr Gly1 5 101936PRTArtificialSynthetic construct
193Gly Lys Tyr Trp Glu Arg1 51949PRTArtificialSynthetic construct
194Arg Asn Gln Tyr Gly Gln His Gln Ser1
519511PRTArtificialSynthetic construct 195Ser Pro Pro Phe Arg Ala
Gly His Val Thr Gly1 5 101966PRTArtificialSynthetic construct
196Val Thr Ala Arg Cys Gln1 519711PRTArtificialSynthetic construct
197Thr Thr Gly Asn Gly Leu Arg Ser Asn Pro Ala1 5
101989PRTArtificialSynthetic construct 198Ser Trp Ala Gln Ala Asn
Pro Gly Gly1 51996PRTArtificialSynthetic construct 199Trp His Ser
Ile Thr Phe1 520010PRTArtificialSynthetic construct 200Lys Thr Lys
Val Gln Ser Ser Asn Pro Ala1 5 102016PRTArtificialSynthetic
construct 201Asp Val Thr Asp Thr Thr1 52029PRTArtificialSynthetic
construct 202Phe Lys Pro Leu Ala Glu Ile Asp Gly1
52037PRTArtificialSynthetic construct 203Lys Asp Val Pro Gly Asp
Arg1 52047PRTArtificialSynthetic construct 204Leu Thr Glu Asp Glu
Asn Gln1 52056PRTArtificialSynthetic construct 205Gly Asn Leu Lys
Pro Asp1 520610PRTArtificialSynthetic construct 206Arg Arg Gly Asp
Met Ser Ser Asn Pro Ala1 5 102077PRTArtificialSynthetic construct
207Lys Asp Val Thr Asp Thr Thr1 52086PRTArtificialSynthetic
construct 208Thr Glu Asp Glu Asn Gln1 52098PRTArtificialSynthetic
construct 209Gly Asn Leu Lys Pro Asp Thr Glu1
521085PRTArtificialSynthetic construct 210Ile Glu Val Lys Asp Val
Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe1 5 10 15Lys Pro Leu Ala Glu
Ile Asp Gly Ile Glu Leu Thr Tyr Gly Ile Lys 20 25 30Asp Val Pro Gly
Asp Arg Thr Thr Cys Asp Leu Thr Gly Glu Asp Glu 35 40 45Gly Asn Gln
Cys Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu 50 55 60Val Ser
Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys65 70 75
80Glu Thr Phe Thr Thr 8521163DNAArtificialSynthetic construct
211accgcgctga ttacctggtc tnnkscgnnk gstnnknnkn nkggcattga
actgacctat 60ggc 6321272DNAArtificialSynthetic construct
212accgcgctga ttacctgggc gvmaccgnnk nnknnkrrcr gcnnkattnn
kggtattgaa 60ctgacctatg gc 7221369DNAArtificialSynthetic construct
213accgcgctga ttacctggtc tccgbstnnk nnknnknnkn nknnkaccgg
cattgaactg 60acctatggc 6921466DNAArtificialSynthetic construct
214tatgaagtga gcctgattag cnnkamsnnk nnkggtnnkn nknnkagcaa
agaaaccttt 60accacc 6621569DNAArtificialSynthetic construct
215tatgaagtga gcctgattag cnnkamsnnk nnknnknnkr gcaacccggc
gaaagaaacc 60tttaccacc 6921672DNAArtificialSynthetic construct
216tatgaagtga gcctgattag cnnkamsnnk nnkggtnnkn nkagcaaccc
ggcgaaagaa 60acctttacca cc 7221763DNAArtificialSynthetic construct
217accgcgctga ttacctggtc tnnkscgnnk gstnnknnkn nkggctgtga
actgacctat 60ggc 6321869DNAArtificialSynthetic construct
218accgcgctga ttacctggtc tccgbstnnk nnknnknnkn nknnkaccgg
ctgtgaactg 60acctatggc 6921972PRTArtificialSynthetic construct
219Ala Cys Cys Gly Cys Gly Cys Thr Gly Ala Thr Thr Ala Cys Cys Thr1
5 10 15Gly Gly Gly Cys Gly Val Met Ala Cys Cys Gly Asn Asn Lys Asn
Asn 20 25 30Lys Asn Asn Lys Arg Arg Cys Arg Gly Cys Asn Asn Lys Ala
Thr Thr 35 40 45Asn Asn Lys Gly Gly Thr Thr Gly Thr Gly Ala Ala Cys
Thr Gly Ala 50 55 60Cys Cys Thr Ala Thr Gly Gly Cys65
7022066DNAArtificialSynthetic construct 220tatgaagtga gcctgatttg
cnnkamsnnk nnkggtnnkn nknnkagcaa agaaaccttt 60accacc
6622169DNAArtificialSynthetic construct 221tatgaagtga gcctgatttg
cnnkamsnnk nnknnknnkr gcaacccggc gaaagaaacc 60tttaccacc
6922272DNAArtificialSynthetic construct 222tatgaagtga gcctgatttg
cnnkamsnnk nnkggtnnkn nkagcaaccc ggcgaaagaa 60acctttacca cc
7222367DNAArtificialSynthetic construct 223ccggtttcag gttaccaatg
ctatamnnmn nmnnmnnmnn mnncagatct atggtggtgc 60gatcgcc
6722479DNAArtificialSynthetic construct 224ccgccaccgg tggtaaaggt
ttctttgctm nnmnnmnnac cmnnmnnskt mnngcaaatc 60aggctcactt catattcgg
7922581DNAArtificialSynthetic construct 225cgccaccggt ggtaaaggtt
tctttcgccg ggttgcymnn mnnmnnmnns ktmnngcaaa 60tcaggctcac ttcatattcg
g 8122685DNAArtificialSynthetic construct 226ccgccaccgg tggtaaaggt
ttctttcgcc gggttgctmn nmnnaccmnn mnnsktmnng 60caaatcaggc tcacttcata
ttcgg 8522726PRTArtificialSynthetic construct 227Met Thr Asn Ile
Thr Lys Arg Ser Leu Val Ala Ala Gly Val Leu Ala1 5 10 15Ala Leu Met
Ala Gly Asn Val Ala Leu Ala 20 2522810PRTArtificialSynthetic
construct 228Arg Leu Asp Ala Pro Ser Gln Ile Glu Val1 5
102295PRTArtificialSynthetic construct 229Ala Leu Ile Thr Trp1
52307PRTArtificialSynthetic construct 230Ile Glu Leu Thr Tyr Gly
Ile1 52314PRTArtificialSynthetic construct 231Thr Thr Ile
Asp12323PRTArtificialSynthetic construct 232Tyr Ser
Ile12339PRTArtificialSynthetic construct 233Thr Glu Tyr Glu Val Ser
Leu Ile Ser1 52346PRTArtificialSynthetic construct 234Lys Glu Thr
Phe Thr Thr1 523585PRTArtificialSynthetic construct 235Ile Glu Val
Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe1 5 10 15Lys Pro
Leu Ala Glu Ile Asp Gly Ile Glu Leu Thr Tyr Gly Ile Lys 20 25 30Asp
Val Pro Gly Asp Arg Thr Thr Cys Asp Leu Thr Gly Glu Asp Glu 35 40
45Gly Asn Gln Cys Ser Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu
50 55 60Val Ser Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn Pro Ala
Lys65 70 75 80Glu Thr Phe Thr Thr 8523611PRTArtificialSynthetic
construct 236Arg Leu Asp Ala Pro Ser Gln Ile Glu Val Lys1 5
102375PRTArtificialSynthetic construct 237Thr Thr Ile Asp Leu1
5238288DNAArtificialSynthetic construct 238ccgccgagcc tgaacgtgac
caaagatggc gatagctata gcctgcgctg ggaaaccatg 60aaaatgcgct atgaacatat
tgatcatacc tttgaaattc agtatcgcaa agataccgcg 120acctggaaag
atagcaaaac cgaaaccctg cagaacgcgc atagcatggc gctgccggcg
180ctggaaccga gcacccgtta ttgggcgcgt gtgcgtgtgc gtaccagccg
taccggctat 240aatggcattt ggagcgaatg gagcgaagcg cgtagctggg ataccgaa
2882396PRTArtificialSynthetic construct 239Lys Asp Val Thr Asp Asp1
52407PRTArtificialSynthetic construct 240Lys Asp Val Pro Gly Asp
Arg1 52418PRTArtificialSynthetic construct 241Gly Asn Leu Lys Pro
Asp Thr Glu1 5
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